The CBEWS-L to be established is envisioned to be information and communication technology (ICT)-based. An ICT4D perspective was therefore taken in conducting this participatory study. Stakeholder mapping, focus group discussions and key informant interviews were used to gather data. Validation was also done through triangulation and post-analysis consultations. The results showed that there are varying sets of capacities and vulnerabilities existing in each community. Moreover, ICT capacities are lacking and are outnumbered by vulnerabilities. Yet, site-specific action points to enhance capacities and resolve vulnerabilities were determined. Still, overall strategies were not uncovered. Furthermore, compounding the capacities and vulnerabilities in each site are indirect factors which, if examined thoroughly, may lead to more complex socio-political issues. It is therefore recommended that in establishing a CBEWS, a comprehensive community risk assessment is first conducted to exhaust all possible action points that can be used in formulating site-specific strategies. Moreover, development of technological solutions must be modular to allow flexibility in accommodating complex community capacities and vulnerabilities.Existing frameworks of community-based early warning systems (CBEWS) lack focus on its actual implementation. Furthermore, they do not describe how a top-down early warning system (EWS) can be transformed into a CBEWS. Hence, to support the establishment of a community-based early warning system for deep-seated landslides (CBEWS-L), this study aimed to assess the capacities and vulnerabilities of five Keywords: Early warning systems; community risk assessment; participatory research; information and communication technology or development; community-based disaster risk reduction and management. Deep-seated landslides are characterised by a slip surface of at least 3 m Kaunda . Their oCompared to shallow ones, deep-seated landslides are said to pose larger risks, as they are more likely to cause disastrous debris flows and landslide dams  which were provided with it. The EWS integrated the monitoring system with community-based monitoring of surficial measurements conducted by the local landslide monitoring committee (LLMC). Local landslide monitoring committee is a volunteer group initially organised by the system implementers. Monitored and analysed data became the basis of early warning information (EWI) sent to the community and the local government units (LGUs).In the Philippines, a monitoring system for deep-seated landslides was initiated in 2008 . Department of Science and Technology – Philippine Institute of Volcanology and Seismology makes use of information and communication technology (ICT) such as computers to collect data from the sites, analyse the risk and generate warning. Early warning information is then sent via short message service (SMS). However, as most communities catered by the EWS are located in far-flung areas, this manner of data transfer and information dissemination results in delay in warning issuance. Hence, there is a need to properly conceptualise the EWS in a manner that appropriately involves the community.Although there is no single definition of the term   also conThe people-centred EWS (PEWS) is related with the community-based early warning systems (CBEWS) concept. The latter is defined as the capability of a community to systematically collect, compile and/or analyse information that is used to provide recommended actions to reduce harm or loss from a hazard event IFRC .Community-based early warning systems can be more cost-effective and politically viable than other strategies such as relocation  in the Philippines  and bottom-up (community-based) approaches  Theory also guided the research. ICT for Development holds that for ICT to be empowering, it should be created through participation, ensuring that all voices are heard and that it leads to the realisation of rights Beardon . Hence, Capacities and vulnerabilities assessment (CVA) was the method used. Capacities and vulnerabilities assessment holds that local communities should undergo ‘self-discovery and self-analysis’ so that the study results could effectively inform the CBEWS planning and decision-making  community diversity (indicated by presence of indigenous groups); (2) site accessibility ; (3) local government unit (LGU) structures/relations (indicated by perceived structure and relations among residents and LGUs); and (4) community resources . Below is a summary of each sample site’s description:Site A – easily accessible through the national highway and has some GSM signal; has reported challenge with LGU structure and relations but belongs to a first-class municipality.Site B – composed of an indigenous group; far from the provincial centre and has limited to no GSM signal; also reported to have negative LGU relations and belongs to a second-class municipality.Site C – composed of an indigenous group; easily accessible through the national highway and is near the municipality centre; has some GSM signal; has good LGU relations and belongs to a fifth-class municipality.Site D1 – far from the municipality centre but has some GSM signal; has good LGU relations and belongs to a second-class municipality.Site D2 – part of the same municipality as D1 and is also far from the municipality centre; has limited to no GSM signal.In each site, the groups that were invited were the barangay council, members of the households at risk  and the LLMC. However, we also included higher tiers of local government disaster risk reduction and management (DRRM) offices  as supporting stakeholders. The number of participants per stakeholder group was based on their availability, but an effort to include people from vulnerable sectors of each community such as youth and older persons was made. The number of participants per study site was as follows: site A – 36; site B – 28; site C – 25; site D1 – 25; and site D2 – 26.To implement the CVA, two workshop activities were used. Firstly, a CVA template adapted from Anderson and Woodrow in Davis et al.  was compIn situ analyses were facilitated using the integrated CVA template explained in the previous section. Gathered data were further content analysed and categorised. The pressure and release (PAR) model , community motivation and relationsphysical resources – all needed ICTs, public utilities, facilities, EWS-L tools and equipment, and financial resourcestrainings and learning resources – resources educating the community on various EWS processes, including landslide sensor data analysisappropriate skills – individual literacy, numeracy, ICT, management and EWS-L technical skills of the communitygovernance and accountability – active involvement of LGUs, coordination across LGU levels and willingness of LGUs to help the community.These categories were lifted from the PAR model, except for the governance and accountability category. Governance and accountability was appropriately coded because the capacities and vulnerabilities falling under this category cannot be related with any of the categories found in the PAR model.Aside from the tabulated data, the participants were able to formulate action points for their capacities and vulnerabilities. In formulating said action points, further capacities and vulnerabilities were revealed as detailed in the following subsections.While the LLMC leader of site A actively participates in the current EWS-L, members are not as participative. According to the LLMC members, compensation for the LLMC can help resolve the issue. For them, local economy is one hindrance to their participation. Specifically, they would rather spend their hours on their livelihoods than volunteer for LLMC .Another action point that the participants think will help improve LLMC performance is to increase community awareness of the landslide risk. According to them, only a few are willing to do volunteer work because most residents are unaware of the deep-seated landslide hazard in their community. They do not fully comprehend the risk they are facing. This was also linked to the low credibility of LLMC and lack of sense of ownership over the EWS-L.On the contrary, the participants attributed their vulnerabilities in terms of physical resources and training and learning resources to lack of government funding. They understand that there must be proper budget allocation for the provision of necessary resources. However, they reported that the overall internal revenue allocation (IRA) of the barangay is limited, thus limiting their DRRM fund.In addition, the specific lack of ICT resources in the community was attributed by the participants to the unavailability of Internet and GSM connectivity provider in the area. To them, as connectivity is weak, it would make no sense to allocate money to ICT resources such as computer.However, according to residents and local officials, they have previously requested private companies to improve connectivity in the area. The community’s request had been rejected because of limited market in the area. Hence, while enhancing connectivity can be an action point to resolve their vulnerability, the participants see such dilemma as beyond their control.Unlike in site A, both LLMC leader and members in site B are well performing. However, the participants, including the LLMC leader and members themselves, believe that this capacity can still be improved. Like in site A, they also think that providing compensation for the LLMC will enhance their performance. According to the participants, most of them cannot sacrifice their livelihood for participating in EWS-L activities such as data gathering. They emphasised that there is limited available livelihood in the area; hence, they must grab every opportunity .On the contrary, the participants believe that to improve their capacity in terms of LLMC participation, more members must be recruited. However, they believe that there is an underlying vulnerability: there are more residents who remain unaware of the EWS-L in their area than those who have previously been engaged by the project. One proof, they said, is the fact that their tribal council is actually not involved in the current system. This makes recruiting active members of the LLMC a challenge.Compounding the problem is the location of houses across the barangay. Some residents, who are willing to participate in the EWS-L, live far from usual place of assemblies. Therefore, the participants said, their desire to keep their communities safe is hindered by the physical inaccessibility of interventions such as trainings.In relation, the participants believe that to make trainings and learning resources effective, poor education in the community must be resolved. Specifically, as literacy and numeracy is a problem among many residents, it is difficult for them to make the most out of trainings and learning resources. Hence, improving basic level education is one action point that they identified. Alternatively, trainings and learning resources must be appropriated to their educational capacity.In terms of physical resources, on the contrary, the participants believe that poor DRRM budgeting in the BLGU and MLGU levels can be resolved by actually increasing government funding for their area. They perceive that the lack of budget at the LGU level is a limiting factor to the official’s capacity to govern them. However, they do not know how their IRA could be increased.Finally, like in site A, construction of cellular sites was identified as one action point that can be done to enhance the ICT resources of the community. However, they too had been rejected by private companies before; hence, they see this action point as highly impossible.For participants in site C, strengthening their indigenous practices can be a primary action point in enhancing their capacities and addressing their vulnerabilities. They believe that most of their capacities are rooted on their culture. If their culture would be upheld, their capacities can be further strengthened .They also believe that although there are now residents who are not part of their indigenous tribe, there should be an effort to at least make them respect their traditions. This way, indigenous DRRM practices and rules can still be effective.pagsigaw (shouting) of warnings from the top of slopes so that the message can be heard by everyone. They still implement this dissemination technique today.However, they maintain that documenting practices is not part of their culture. They said that they are able to preserve their DRRM practices even without documentation. One example is their practice of Yet, the provincial LGU reiterated that although cultural practices are an advantage, it is also good to have written rules. Plans and policies are important in DRRM practice. They also said that to enhance planning and policy-making in the barangay, the provincial and municipal LGUs can provide trainings to the barangay LGU.According to the participants, assistance to the local authorities in terms of planning and policy-making is both doable and advantageous. Firstly, site C is located near the municipality centre; hence, assistance can easily be given to local authorities. Second, they are convinced that plans and policies can enhance government funding. When there is proper funding, they believe that training and physical resources can easily be provided to them.On the contrary, unlike in sites A and B, residents of site C are highly aware of the landslide hazard they are facing. Site C is situated in a mountainous region where shallow landslides frequently happen. Hence, encouraging people to participate in the EWS-L is, according to the participants, easy. They added that perhaps further provision of trainings would help improve this capacity.However, although highly aware of the landslide risk, the participants expressed challenge with addressing the lack of computer skills among residents. They understand that the EWS-L uses ICTs and they also believe that ICTs make the system operation easier. However, they reported that acquiring computer skills can be a challenge especially because most site C residents are elderly. At their age, they are unwilling to adapt to new technologies. A solution, according to the participants, will be to engage younger residents.Furthermore, they believe that one solution to the poor Internet and GSM connectivity in the area is to strengthen governance. They believe that engaging private entities will be easier if LGUs will lead the action.Observable landslide indicators are present across the deep-seated landslide-prone area in site D1. Most residents are therefore aware of the presence of such hazard. This is what site D1 considers as one of the reasons why the LLMC is well performing. According to the participants, it is therefore a must that PHIVOLCS continues its efforts in educating the residents .On the contrary, conflicts among residents are easily resolved. The participants said that they are all God-loving. Hence, it is easy to make peace within the community.However, unlike enhancing social capacities and resolving social vulnerabilities, site D1 feels that addressing their insufficient physical resources is a huge challenge. For one, they cited the issue of low IRA, which they claimed not within their control. However, they think that educating government officials might help with funding.From another perspective, local authorities of site D1 believe that enhancing DRRM funding in the barangay can be done through trainings. Specifically, the provincial and municipal governments are planning to conduct training on DRRM policy-making for both sites D1 and D2 because they belong to the same municipality. According to the PLGU and MLGU, trainings on policy-making will enable the BLGU to allocate its DRRM fund appropriately.In addition, barangay DRRM policies are also foreseen to be helpful in enhancing the dissemination skills of the community. For instance, SMS is currently their means of communication but GSM signal is weak in the site. They believe that the use of siren or flags will be helpful. To do so, they said they need a resolution that standardises the use of these channels – how long is the alarm for each deep-seated landslide alert level, which flag signals which alarm?Participation in the EWS-L and in decision-making is apparent in site D2 through active LLMC and open general assemblies. However, the participants feel that these capacities can still be enhanced. Particularly, they believe that the conflict between their livelihood and volunteer work needs to be resolved .According to the participants, the need to attend to their livelihoods hinders scheduling trainings and encouraging attendance which they believe would facilitate the CBEWS-L realisation. Specifically for site D2, harvest season comes every April and September. Hence, EWS-L processes, including capacity-building activities, are disrupted during these periods. A solution, according to the participants, is to take turns in performing surficial data gathering and to request PHIVOLCS to schedule the trainings accordingly. This would entail clarifying roles within the organisation and strengthening coordination with PHIVOLCS.In relation, they also feel that although the LLMC is active, engaging other residents can be helpful to them. Specifically, like in D1, active participation of LLMC is attributed to their awareness of the landslide hazard. Hence, according to the participants, if more residents are made aware, more support for the LLMC members can be gained.On the contrary, one social characteristic of the community which the participants think will help resolve their vulnerabilities is their being indigenous peoples (IP). They believe that filling the gaps in their physical resources can be addressed by capitalising on their indigenous identity. According to them, they can easily request for assistance from the government because IPs are given priority in their region.Alternatively, they see the need to strengthen DRRM policies at the barangay level to help them gain financial support from the provincial and municipal government. However, participants from the barangay LGU themselves expressed insufficient of skills in policy-making, hindering the realisation of proper budgeting and later provision of necessary physical resources.batingaw or makeshift bells in disseminating deep-seated landslide alerts. Hence, with such expression of capacity, the proposed training on DRRM policy-making by the provincial and municipal governments for site D2 is seen not as a first step but as another step forward towards capacity improvement.But this is not to say that the BLGU has not done DRRM-related policies at all. In fact, the participants emphasised that they now have a resolution standardising the use of The site-specificity of capacities and vulnerabilities as posited by Davis et al.  is apparFor example, resolving challenge with literacy and numeracy has only been brought up in site B. This is primarily because it is only in site B where marginalisation is rampant, as described by the participants themselves. On the contrary, upholding cultural practices is an action point that has been uniquely identified in site C. Although three study sites  were selected for having indigenous residents, it is only in site C where traditional practices had been maintained. Evidently, differences in site conditions affected variations in capacities, vulnerabilities and corresponding action points.However, although the overall set of capacities and vulnerabilities in one site may differ from the other, certain existing capacities or vulnerabilities were observed in more than one site. For example, all sites mentioned the following capacities:availability of evacuation centresEWS trainingsskills in surficial data gathering.The last two items are apparent because these are the main themes discussed during past project capacity-building activities. Other capacities and vulnerabilities existing in more than one study site are as follows:active LLMC leaders or memberslack of ICT materialspoor ICT infrastructurelack of ICT skillsvarying challenges in DRRM practice at the barangay level.Apparently, organisational, material and governmental challenges are common in all study sites. It is evident from these results that although the study sites are unique, certain characteristics can be similar. Hence, although it is true that there is no single realising framework for establishing an EWS  – influences social relations because the need to attend to livelihood hinders LLMC and community participation in the EWS-L, such as in the case of sites A, B and D2.Education (basic literacy and numeracy) – a challenge in providing trainings and learning resources in site B.Culture (indigenous knowledge and practices) – how DRRM practices are established in site C, hence hindering the institutionalisation of the CBEWS-L.Awareness (risk and project awareness) – hinders the credibility and participation of LLMC in site A, and facilitates local participation in sites C, D1 and D2.Physical environment (location of residence and access to facilities) – like local economy, this influences LLMC and community participation specifically in site B.Government funding (IRA or DRRM fund) – dictates provision of physical resources and training in site A, affects governance and accountability in Site B and determines provision of physical resources in site D1.Local investment (private investment to infrastructure) – lack of network infrastructure hinders availability of ICT materials in sites A, B and C.Conditions falling under these categories do not directly facilitate or hinder the CBEWS-L realisation; rather, they influence the facilitating or hindering factors per se. This finding agrees well with the progression of vulnerability into dynamic pressured and root causes, posited by Wisner et al. .Therefore, it is recommended that while action points can be shared among sites, strategies should be site-specific. For instance, incentivising the LLMC in sites A, B and D2 to enhance LLMC and community participation may yield varying results. An incentive system for site A, where risk awareness is a problem, might still not encourage residents to become LLMC members. Similarly, incentives for site B may be more effective because they are more aware of their risk; however, the issue of education will still hinder the capacity-building needed for LLMC members. On the contrary, incentives might serve as a cherry on top to the already well-performing LLMC of site D2.In other words, the combination of action points, their order and manner of implementation is highly dependent on present and pressing community conditions, especially because some of the said community conditions are spatially distant or are not within the control of the community, the local authorities or PHIVOLCS.To consider all possible factors, it is recommended that a more systematic use of the PAR model is done. This means that communities must first be well oriented with the notion of progression of capacities and vulnerabilities. Then, they should be able to identify clear linkages among factors that may facilitate or hinder the CBEWS-L realisation. In doing so, even the root causes, which are deemed systemic, can be made known.Contrast among the sites’ capacities and vulnerabilities is most apparent in terms of social relations and governance and accountability. This indicates that establishing a CBEWS-L must not only be technological, but should also be both social and political.Previous literature states that the effectiveness of an EWS (or any DRRM effort for that matter Allen ) cannot Therefore, aside from uncovering social and political capacities and vulnerabilities, a socio-technical approach in strategy-building must be taken as well. In EWS, a socio-technical approach entails the use of technology which is appropriate to psychosocial site conditions (Karnawati et al. In the case of this study, the possibility of using technological solutions in the CBEWS-L was given emphasis. However, it has been clear in the results that there are several gaps in community resources in terms of ICTs. These gaps are not just limited to material resources, but to the willingness, learning resources and skills of communities, compounded by political conditions as well.Following the ICT4D perspective Beardon , it is eHence, it is recommended that the development of technologies for the CBEWS-L should be made modular. A modular approach and phased process allows for emergence of new requirements (Waller et al. The complexity of capacities and vulnerabilities in the study site necessitates the participation of multiple stakeholders in implementing the CBEWS-L. For instance, funding and provision of other services and resources are deemed by the communities as dependent mostly on the government. Moreover, local investment, which is highly dependent on private sectors, was also seen as a factor for CBEWS-L success, especially in terms of communication utilities.These findings highlight the need for linkages and collaborations where resources are insufficient, as suggested in literature Parkash . As seenAlternatively, previous literature indicates that the use of simpler technologies where resources are limited can be successful (Abon et al. Therefore, a multi-stakeholder approach to the CBEWS-L implementation is necessary. Furthermore, the multiplicity of stakeholders to be engaged must be ensured throughout the CBEWS-L realisation process – from the design of the system, its development and its actual implementation.In introducing this study, we argued that establishing a CBEWS-L necessarily includes the four PEWS elements UNISDR . We alsoThe results of such assessment now further prove the importance of risk knowledge. The lack of risk and EWS-L awareness among locals was deemed by the research participants as a hindrance to the CBEWS-L vision. Awareness was found to be an underlying factor to social relations. Particularly, lack of awareness among locals results in low participation in the EWS-L.As mentioned, deep-seated landslides are characterised by very slow movements Kaunda . Hence, The nature of deep-seated landslides therefore further highlights the necessity of tackling risk knowledge prior to the CBEWS-L establishment. To date, no comprehensive risk knowledge generation has been done in any of the EWS-L sites; hence, a community risk assessment is highly recommended. Moreover, to uncover root causes, the PAR model should be used as a conceptual framework for future studies.The study was qualitative and participatory, and the unit of analysis is the community. Hence, the expected results are site-specific and not generalisable. While this is the case, five case studies were purposively sampled to cover different situations of communities in the Philippines. Results showed similarities and differences across all five cases. Quantitative data were not gathered; hence, the study can be validated or falsified by more experimental designs.Moreover, the research results only described the communities’ capacities and vulnerabilities, but did not indicate actions that the communities must take. In addition, while the research adhered to the PEWS framework UNISDR , the maiThrough the use of participatory assessments, capacities and vulnerabilities of five barangays in establishing EWSs for deep-seated landslides were identified in our study, along with action points to address them. Identified situations were highly site-specific. However, similarities in themes allow for strategic solutions in establishing CBEWS-L. Solutions were only identified, and in-depth planning of strategies is still recommended.While the initial focus of the study is to understand the capacity of the community of operating EWS-L consisting of ICT, framing the CBEWS-L realisation as both a social and political endeavour is necessary. Not only should the technological capacities and vulnerabilities of the study site be examined, but underlying factors should be considered as well.A more comprehensive community risk assessment that uses the PAR model is still needed. Furthermore, contin-uously using a participatory approach to the CBEWS-L implementation is necessary so as to include the multiple stakeholders who may be key to the success and sustainability of the system.


We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells. KCNQ genes, mediate “M-currents”, which have been implicated in the regulation of neuronal excitability by G protein-coupled receptor agonists cyt); hence, mechanisms that reduce [Ca2+]cyt will tend to relax smooth muscle. One such mechanism, supported by results described above, is an increase in outward potassium current via Kv7.5 channels, which would result in membrane hyperpolarization and consequently reduce influx of Ca2+ via L-type voltage-sensitive Ca2+ channels cyt . The relCa2+]cyt  suggestss/cAMP/PKA pathway is mediated by phosphorylation of S53 on the amino-terminal region of the Kv7.5 α-subunits. Mutation of S53 to either alanine (A) or aspartic acid (D) prevented forskolin-induced enhancement of the current. The S53A construct is expected to be non-productive in terms of PKA-dependent responses because it cannot be phosphorylated at that site  and alanine does not confer the negative charge that would result from phosphorylation of a serine at that site. In contrast, substitution at the serine site with a negatively charged aspartic acid residue  might be expected to serve as a phospho-mimic, as has been observed for other ion channels , where G is conductance, maxG is a maximal conductance, V0.5 is the voltage of half-maximal activation and k is the slope factor. Each conductance curve for each experiment was normalized by maxG from the Boltzmann fit and G/maxG curves from all corresponding individual experiments were averaged to produce conductance plots presented in the figures and fitted to the Boltzmann distribution without any constraints. Voltage dependence of endogenous current activation in HASMCs was determined by converting steady-state end pulse current to conductance, according to the equation: G = I/(V − KE), where I is the current amplitude, V is the step potential and KE is the reversal potential for potassium (−101 mV). Voltage-dependence of wild-type and mutant Kv7.5 channel activation was estimated as described previously  from thet-test. For comparisons between two independent groups, t-test or the t-test followed by the Mann-Whitney rank-sum test was used. Differences among multiple treatment groups were assessed by analysis of variance (ANOVA) followed by a Holm-Sidak post hoc test or ANOVA on ranks followed by multiple comparisons vs. control group (Dunn method). Differences were considered statistically significant with p values ≤ 0.05.Data were analyzed using SigmaPlot  and are expressed as mean ± standard error of the mean (S.E.). Parameters measured before and after treatments were compared using a paired Student’s KCNQ5 cDNA  was a generous gift from Dr. T. Jentsch at the Max-Delbrück-Centrum for Molecular Medicine .Cell culture media were from MediaTech . Fetal bovine serum was from Atlanta Biologicals. Antibiotic/antimycotic solution, Formoterol fumarate, 3-Isobutyl-1-methylxanthine (IBMX) were from Sigma-Aldrich. Amphotericin B, H-89 dihydrochloride were from Calbiochem. Forskolin was from Tocris Bioscience . Retigabine was from Alomone Labs . The AdEasy Adenoviral Vector System was from Stratagene. The QuikChange Site-Directed Mutagenesis Kit was from Agilent Technologies . The human


The total bacterial community plays an important role in aquatic ecosystems. In this study, bacterial communities and diversity along the shores of the Upper Gulf of Thailand were first characterized. The association between bacterial communities and types of land use was also evaluated.5, were significantly different when grouped by land use. The bacterial communities were mainly determined by salinity, total N, and total P. The species richness estimators and OTUs were positively correlated with turbidity. The top ten most abundant phyla and genera as well as the distribution of bacterial classes were characterized. The Proteobacteria constituted the largest proportions in all sampling sites, ranging between 67.31 and 78.80%. The numbers of the Marinobacterium, Neptuniibacter, Synechococcus, Candidatus Thiobios, hgcI clade (Actinobacteria), and Candidatus Pelagibacter were significantly different when grouped by land use.The bacterial communities and diversity of seawater in the Upper Gulf of Thailand, with regard to types of land use, were first revealed by using Illumina next-generation sequencing. A total of 4953 OTUs were observed from all samples in which 554 OTUs were common. The bacterial communities in sampling sites were significantly different from each other. The run-off water from three types of land use significantly affected the community richness and diversity of marine bacteria. Aquaculture sites contained the highest levels of community richness and diversity, followed by mangrove forests and tourist sites. Seawater physicochemical parameters including salinity, turbidity, TSS, total N, and BODProteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Verrucomicrobia, Euryarchaeota, Planctomycetes, Firmicutes, Deep Sea DHVEG-6, and Marinimicrobia were the most and common phyla distributed across the Upper Gulf of Thailand.Type of land use significantly affected bacterial communities and diversity along the Upper Gulf of Thailand. Turbidity was the most influential parameter affecting the variation in bacterial community composition. Salinity, total N, and P were the ones of the important factors that shaped the bacterial communities. In addition, the variations of bacterial communities from site-to-site were greater than within-site. The  It is a semi-enclosed tropical marine embayment which is surrounded by the land masses of Thailand, Cambodia, Vietnam, and Malaysia . The coa4 , human dTo explore the total bacterial community in the environment, high-throughput next-generation sequencing (NGS) technology of the taxonomically informative 16S rRNA gene provides the most powerful approach because it enables the classification of individual reads to specific taxa . The conIn this study, we investigated 1) the marine bacterial communities at nine sites along the shores of the Upper Gulf of Thailand by using Illumina NGS of the V4 variable region of 16S rRNA gene; 2) the association between bacterial community structures and three types of land use including mangrove forests, tourist sites, and aquaculture sites; 3) effect of seawater physicochemical parameters on the abundance of specific taxa; and 4) the correlation between seawater physicochemical parameters and types of land use.5) at nine sampling sites are shown in Table 5 values of all sites ranged between 0.90 ± 0.00 and 3.76 ± 0.23 mg/l. Site B (mangrove forest at Kungkrabaen Bay) had the highest BOD5 value, differing significantly from that of other sites. On the contrary, sites F (aquaculture site at Donhoylhod) and I (tourist site at Wanakorn Beach) had the lowest BOD5 values.Nine sampling sites in seven provinces along the shores of the Upper Gulf of Thailand, over a distance of approximately 769.97 km, are shown in Table P = 0.27, 0.35, and 0.13). On the contrary, seawater temperature, % NaCl, turbidity, TSS, total N, and BOD5 were significantly different when grouped by land use . Turbidity, TSS, and total N of aquaculture sites were highest, differing significantly from those of other types of land use. BOD5 value of mangrove forests was highest, differing significantly from that of tourist sites. Aquaculture sites had the lowest values of % NaCl that were significantly different from those of other types of land use.In this study, the results in Table P = 0.02 and 0.01) (Table P = 0.00 and 0.00). The bacterial community diversity of aquaculture sites was significantly highest, followed by mangrove forests and tourist sites, respectively . After tag merge and quality control, a total of 2,425,463 clean tags (97.85% of raw reads) were obtained. After that, potential chimera tags were removed with the UCHIME algorithm, resulting in a total of 2,177,667 taxon tags. The tags with ≥97% similarity were grouped into the same operational taxonomic units (OTUs). A total of 4953 OTUs were observed from all samples, with a mean Good’s coverage of 99.00 ± 0.00%. ACE (abundance-based coverage estimator) and Chao1 that represent richness as well as Shannon-Weaver and Simpson that indicate diversity were analyzed  of site F (aquaculture site at Donhoylhod) was highest, followed by sites A (mangrove forest at Black Sand Beach) and E (aquaculture site at Angsila old market), respectively. The bacterial richness of site I (tourist site at Wanakorn Beach) was lowest. Higher Shannon-Weaver and Simpson indices indicate greater bacterial diversity. The highest values of both indices were found in site G (aquaculture site at Bangtaboon Bay), followed by sites F (aquaculture site at Donhoylhod) and C (tourist site at Suanson Beach), respectively. The lowest values were found in site D (tourist site at Pattaya Beach).Rarefaction analysis was used to standardize and compare taxon richness among samples and to identify whether the samples were randomly selected. According to the rarefaction curves of samples , Cyanobacteria (2.64–15.16%), Actinobacteria (1.41–11.68%), Verrucomicrobia (0.30–1.89%), Euryarchaeota (0.24–1.47%), Planctomycetes (0.19–1.04%), Marinimicrobia (0.05–0.92%), Firmicutes (0.02–0.41%), Deep Sea Hydrothermal Vent Group 6 , and others (1.09–1.25%). The distribution of bacterial classes in each sampling site is shown in Fig. Holophagae, Anaerolineae, and Chloroplast were more abundant than others. The Bacilli, OM190, Sphingobacteriia, Acidimicrobiia, and Verrucomicrobiae were the predominant classes in site B (mangrove forest at Kungkrabaen Bay). The greatest abundance of the JdFBHP3 was found in site C (tourist site at Suanson Beach). The Mollicutes, γ-Proteobacteria, and Opitutae were the predominant classes in site D (tourist site at Pattaya Beach). Site E (aquaculture site at Angsila old market) had the most abundance of the γ-Proteobacteria. Site F (aquaculture site at Donhoylhod) harbored the high numbers of the Bacteroidia, Epsilonproteobacteria, Nitriliruptoria, and Clostridia. Site G (aquaculture site at Bangtaboon Bay) had several most-abundant classes such as the Chloroflexia, Spartobacteria, Thermoleophilia, Chlorobia, and Planctomycetacia. The unidentified Marinimicrobia and unidentified Cyanobacteria were the most abundant classes in sites H (mangrove forest at Pranburi forest park) and I (tourist site at Wanakorn Beach), respectively.The top ten most abundant phyla among nine sampling sites were in different patterns, as depicted in Fig. Marinobacterium was presented in four sites including E (aquaculture site at Angsila old market), H (mangrove forest at Pranburi forest park), G (aquaculture site at Bangtaboon Bay), and A (mangrove forest at Black Sand Beach), which were not significantly different from each other. Site D (tourist site at Pattaya Beach) had the greatest number of the Neptuniibacter, differing significantly from that of other sites. Site I (tourist site at Wanakorn Beach) had the highest abundances of the Synechococcus and Candidatus Pelagibacter which were significantly different from those of other sites. The numbers of the Candidatus Actinomarina, Candidatus Thiobios, Vibrio, and Marinomonas were significantly highest in sites B (mangrove forest at Kungkrabaen Bay), G (aquaculture site at Bangtaboon Bay), C (tourist site at Suanson Beach), and E (aquaculture site at Angsila old market), respectively.The top ten most abundant genera present in each sampling site are shown in an Additional file P < 0.001). Moreover, the variations in community composition among groups and within groups were evaluated by analysis of similarity (ANOSIM) and multi-response permutation procedure (MRPP). The results of both methods indicate that there were significant differences when comparing microbiota by sampling site (P < 0.05), and the variations of inter-group were larger than those of inner-group (r = 1). The unweighted-pair group method with arithmetic mean (UPGMA) dendrogram of the relative abundance at the phylum level depicted in Fig. The ordination of samples by principal coordinate analysis (PCoA) shown in Fig. α-Proteobacteria and Flavobacteriia were positively associated with % NaCl  and negatively associated with TSS . The γ-Proteobacteria were also positively associated with % NaCl  and negatively associated with total P . The members of β-Proteobacteria, unidentified Proteobacteria, and Actinobacteria were positively associated with total N  and negatively associated with % NaCl . The Cyanobacteria were positively associated with total P  and negatively associated with BOD5 . The Acidimicrobiia were positively associated with total N  and negatively associated with seawater temperature . The δ-Proteobacteria were positively associated with BOD5  and negatively associated with total N . Moreover, in this study we also found that the numbers of the Marinobacterium, Neptuniibacter, Synechococcus, CandidatusThiobios, hgcI clade (Actinobacteria), and Candidatus Pelagibacter were significantly different when grouped by land use . The numbers of Synechococcus and Candidatus Pelagibacter were significantly higher in tourist sites than those in other types of land use, whereas Candidatus Thiobios was only one genus whose number was significantly higher in aquaculture sites than that in other types of land use  and G (aquaculture site at Bangtaboon Bay) was different from that of other sites. This may be affected by the physicochemical factors that shaped the bacterial communities of those two sites which ranked first and second in turbidity, TSS, and total N. Moreover, the proportions of the Proteobacteria were highest in all sampling sites, followed by the Bacteroidetes and Actinobacteria. This result corresponds with that of Suh et al. [Proteobacteria was the dominant phylum in seawater from Gosung Bay, South Korea, followed by the Bacteroidetes and Actinobacteria. Similarly, most of the bacterial sequence reads in marine sediments from Jeju Island, South Korea, were also associated with the Proteobacteria and Bacteroidetes, followed by the Actinobacteria, Acidobacteria, and Firmicutes [In this study, a total of 4953 OTUs were observed from all samples. Indeed, the amount of the bacterial OTUs was not necessarily correlated with location. The amounts of the bacterial OTUs in samples greatly varied depending on several physicochemical and environmental factors , 30. Othh et al.  who repormicutes .Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Verrucomicrobia, Euryarchaeota, Planctomycetes, Firmicutes, Deep Sea DHVEG-6, and Marinimicrobia were the most and common phyla distributed across the Upper Gulf of Thailand.This is the first report of the bacterial communities and diversity associated with seawater along the Upper Gulf of Thailand that was categorized into three types of land use including mangrove forests, tourist sites, and aquaculture sites. The run-off water from each type of land use significantly affected the community richness and diversity. The highest community richness and diversity were obtained from aquaculture sites, followed by mangrove forests and tourist sites. Turbidity was the most influential parameter affecting the variation in bacterial community composition. Salinity, total N, and P were the ones of the important factors that shaped the bacterial communities in near-shore seawater from the Upper Gulf of Thailand, whereas pH and seawater temperature less affected the bacterial communities. In addition, the variations of bacterial communities from site-to-site were greater than within-site. The Seawater was sampled on 10th November and 1st December 2018 at nine sites in seven provinces along the shores of the Upper Gulf of Thailand, over a distance of approximately 769.97 km  were measured within 48 h by using a pH meter (Metrohm 827 pH lab), a hand refractometer (Atago N-1E), and a turbidimeter (HACH 2100P), respectively. BODociation  by using®Water DNA kit , according to the manufacturer’s instruction. The V4 variable region of the 16S rRNA gene was amplified by using the 515F and 806R specific primer set with the barcodes [® High-Fidelity PCR Master Mix . The PCR products were purified using a Qiagen gel extraction kit . The libraries were generated with TruSeq® DNA PCR-Free sample preparation kit , and analyzed by HiSeq2500 PE250 sequencing system , according to the manufacturer’s instructions. Negative controls (sterile water) were carried out through amplification and sequencing. Data was returned as fastq files and deposited in the Sequence Read Archive of the National Center for Biotechnology Information under BioProject accession number PRJNA530863 (SRA: SRP190963).Seawater samples were prefiltered through sterile Whatman no. 2 filter papers to remove suspended particles and the filtrate was subsequently filtered through 0.2 μm sterile cellulose nitrate membrane filters  . DNA wasbarcodes , 35. PCRPaired-end reads were merged by using the FLASH program (V1.2.7) . QualityAlpha diversity, including community richness (Chao1 and ACE estimators), community diversity (Shannon and Simpson indices), and index of sequencing depth (the Good’ coverage) as well as rarefaction data were calculated by using the QIIME software (V1.7.0) and displayed by the R software (V2.15.3). PCoA was performed to obtain principal coordinates and visualize complex, multidimensional data, which was then displayed by the WGCNA, stat, and ggplot2 packages in the R software (V2.15.3). The UPGMA clustering was performed as a type of hierarchical clustering method to interpret the distance matrix using average linkage and conducted by using the QIIME software (V1.7.0). The nonparametic method, ANOSIM, was conducted to determine whether the bacterial community structures significantly differ among groups and within groups. MRPP was calculated with the R software (V2.15.3). AMOVA was performed using the Mothur software (V1.36.1). Seawater parameters, alpha diversity indices, and physicochemical parameters of each type of land use were subjected to an analysis of variance (ANOVA) using Tukey’s test. Spearman rank correlation was used to analyze the effect of seawater physicochemical parameters on the bacterial communities. ANOVA and Spearman rank correlations were performed with the SPSS statistical software (V19.0) . All statistical analyses were evaluated at α = 0.05.Additional file 1: Figure S1. Rarefaction curves of observed species number from three samplings of each sampling site. Site A, mangrove forest at Black Sand Beach; B, mangrove forest at Kungkrabaen Bay; C, tourist site at Suanson Beach; D, tourist site at Pattaya Beach; E, aquaculture site at Angsila old market; F, aquaculture site at Donhoylhod; G, aquaculture site at Bangtaboon Bay; H, mangrove forest at Pranburi forest park; I, tourist site at Wanakorn BeachAdditional file 2: Figure S2. OTUs flower analysis of sampling sites.Additional file 3: Table S1. Top ten most abundant genera present in each site. All sites were sampled in triplicate (n = 3). *Values are the means from three samplings ± standard deviations. **Values with the same letters within a column are not significantly different according to Tukey’s test.Additional file 4: Table S2. Top ten most abundant genera present in each type of land use. *Values are the means of three samplings from each location ± standard deviations. **Values with the same letters within a column are not significantly different according to Tukey’s test. ***p-values < 0.05 are considered significant.


This transport process has similarities to enzyme kinetics: both have saturation kinetics, the Km being the concentration at which half maximal transport velocity, Vmax obtained, is a measure of apparent affinity of ligand for the transporter  was initially constructed to rationalize and explain a few readily quantifiable and reproducible experimental findings. The AATM, as originally conceived, consists of single specific binding site centrally situated within the cell membrane. The site alternately faces inwards and outwards. During the inversion process, the site can transport sugar across the membrane, and dissociation into the alternate bathing solution results in net transport. Return of the empty site reinitiates the transport cycle as illustrated in Fig. ter Fig. B. The pr et al. An important kinetic finding, which implied the existence of mobile components within the passive glucose transporter, was the demonstration of countercurrent overshoot—uphill transport of isotopically labelled glucose into red cell cytosol, driven by a downhill counterflow of unlabelled sugar from the cytosol to the external solution  than of net influx and the Km for the process also (≈10×) higher than the Km for net glucose influx . Using several different protocols, it was shown that the glucose affinity on the export inside site of the erythrocyte transporter was approximately tenfold lower than for the glucose import site on the outside  and outwards (Cin → GCin → GCout → Cout) short-circuits the slow path of vacant-carrier outward transits (Cin → Cout) or (Cout → Cin)  Fig. A, where Km(exchange) than the Km(net influx)  is mainly determined by the low-affinity inside site with a higher Km(in), whereas the Km(net influx) is determined only by the high-affinity external facing site  is again determined from the sugar concentration at which the unidirectional rate is half maximal.As well as having a higher rate, the glucose exchange process also has a higher ux) Fig. B. This dKDi is the dissociation constant of ligand for the transporter site at any face i  is Li/KDi, superscript “i” refers to the internal or external solution concentration of transported ligand  .In the non-ideal case this may be expressed asAt equilibrium, since the activities and chemical potentials of a substance must be uniformly distributed between all connected phases i and j, it follows thatxBi ≠ xBj, it follows that sinceIf at equilibrium Cin/Cout = 10, then γCin/γCout = 1/10.In the case of a mobile carrier, as the activities of all mobile components must be the same at equilibrium, then if  et al.It has been asserted that no energy difference is implied in the asymmetric distribution of carrier chemical potentials at equilibrium even though “the activities or concentrations or states 1 and 4 do not have to be equal”  > Km(out) , equivalent to ([Cout] + [GCout])/([Cin] + [GCin]), describes the ratio of total Cout and total Cin as a function of transported ligand concentration, [S]. This assumption implies that the sum of the carrier forms equilibrate between each side  helix N-domain is relatively immobile in comparison with the six TM C-terminal domain which contains segments that bend inwards and outwards during inward–outward facing transition as a result of discontinuities in TM7 and TM10 /Km(out) ≈10 is asymmetric and with the T295M mutation the Km(in)/Km(out) ≈0.6 /Km(out))/(Vmax(in)/Km(in)) from ≈1 in control to ≈0.3  containing seven transmembrane helices can adopt conformations that match the open in, occluded and open out conformers demonstrated by crystal structures  suggests that substrate binding altered the number of FRET states from two in the apo state to one, stabilizing the holo bound conformation in an inward facing state  in AThese results with SWEET transporters may not fully reflect the conformational changes that occur in GLUTs which are much larger and have more complex extramembranous structures, particularly in the cytosolic domains. Much remains to be demonstrated with atomistic and perhaps also coarse-grained Martini MD simulations of sugar transport before an informed view regarding allosteric or induced-fit interactions of the transporter with transported and non-transported inhibitory ligands can be assigned with confidence. Nevertheless, the observed kinetics and thermodynamics of glucose transport via GLUT1 are clear enough to show that they are inconsistent with the AATM and thus some form of staged diffusion is currently a more satisfactory description of both control GLUT1 transport and for several GLUT1DS mutations.


Here, we present a strategy for the rapid, accurate and high-throughput discovery of in vitro kinase substrates using quantitative proteomics. Using 385 purified kinases , we identified a total of 175,574 potential direct kinase substrates. In addition, we identified novel kinase groups, such as one group containing 30 threonine-directed kinases and another containing 15 serine/threonine/tyrosine kinases. Surprisingly, we observed that the diversity of substrates for tyrosine kinases was much higher than that for serine-threonine kinases.Kinase networks are important for cellular signal transduction. Despite tremendous efforts to uncover these signaling pathways, huge numbers of uncharacterized phosphosites still remain in the human proteome. Because of the transient nature of kinase-substrate interactions  Based on the results from human genome project, at least 518 protein kinases are considered to be encoded in human genome2, and at least 70% of all human proteins are phosphorylated by these kinases4. This indicates that each protein kinase involves roughly hundreds of phosphorylation events, and the phosphorylation network formed by them must be extremely complicated.Reversible protein phosphorylation catalyzed by both protein kinases and phosphatases plays essential roles in cellular signal transduction that controls a variety of cellular functions5, and inhibitors of oncogenic kinases have been extensively developed for cancer therapy since the success of imatinib for Bcr-Abl-positive chronic myeloid leukemia (CML) patients. However, although genomics- and proteomics-based approaches have been applied to characterize kinases8, we still lack basic information on many kinases, such as their substrates, phosphorylation motifs and molecular functions, and more information is required to unveil the entire network of protein phosphorylation.It is well known that kinase-mediated phosphorylation signals are involved in various diseases such as cancer, and often cause the disease itself or drive its progression. Large-scale cancer genome sequencing projects have been conducted to detect mutationally activated proteins, including kinases, in cancer signaling pathways9, immobilized metal ion affinity chromatography (IMAC)11 and metal oxide chromatography with enhancers15. Many biological applications of these technologies have been reported, e.g., to study the signal transduction of epidermal growth factor16, cell cycle regulation17, embryonic stem cell differentiation18 and tissue-specific phosphorylation events19, resulting in the identification/quantification of tens of thousands of phosphosites. Thus far, over 100,000 phosphosites in the human proteome have been registered in public databases such as UniProtKB20, PhosphoELM21, PhosphoSitePlus22, PHOSIDA23 and HPRD24. Although recent advances in MS-based phosphoproteomics have enabled identification of more than 10,000 sites by a single LC/MS/MS measurement25, the nature of the exact kinases responsible for these sites largely remains to be elucidated. Moreover, even at the protein level, less than 40% of phosphoproteins can be mapped to the KEGG pathway database26, and the functional roles of the phosphorylated sites are still unclear in many cases27.In recent years, mass spectrometry (MS)-based phosphoproteomics have been improved, especially phosphopeptide enrichment approaches, such as immunoprecipitation with anti-phosphotyrosine antibodiesin silico prediction of kinase-substrate relationships. On the basis of limited dataset of in vitro kinase assay, various computational tools for prediction of responsible kinases or potential substrates, such as Scansite28 and NetPhorest29, have been developed to date. In some cases, public datasets on protein-protein interaction have been integrated in some computational tools for kinase prediction from queried phosphosites32. Many in silico prediction tools for kinase-substrate relationships have been developed33, however, a bottleneck in these tools is the absence of a sufficient experimental kinase-substrate dataset to provide phosphorylation motifs of an individual protein kinase.It is considered that in general each kinase prefers to phosphorylate the substrates with particular sequence, namely, a phosphorylation motif, which often has been utilized for 34 or a position scanning peptide library35 have been employed to extract phosphorylation motifs rapidly38. Mok et al. have reported large-scale analysis of yeast phosphorylation motifs using peptide library screening and identified motifs for 61 kinases39. Newman et al. have reported 300 phosphorylation motifs for 289 kinases by in vitro kinase profiling using a protein array, although phosphosites were not experimentally identified40. These array-based approaches allow a high-throughput profiling of substrate preference for each kinase in a large scale. Regarding MS-based approaches with a biological sample as a substrate source, Huang et al. identified PKA and PKG substrates from rat uteri by using an in vitro kinase assay followed by a phosphoproteomics approach41; 61 and 12 in vitro substrates as well as consensus phosphorylation motifs were found for PKA and PKG, respectively, and found that some of the in vitro substrates were potential physiological substrates. Zhang et al. also reported a solid-phase approach for the identification of in vitro CK2 substrates using immobilized proteins from a cell extract as a substrate source42, and suggested the method may reduce effects of intrinsic active kinases compared with solution-based approach. Kettenbach et al.43 and Douglas et al.44 investigated in vitro kinase substrates using digested peptides from a cell lysate as a substrate source and identified hundreds to thousands of in vitro substrates of each kinase. Furthermore combination use of in vitro assay and in vivo phosphoproteome analysis was utilized to identify physiologically relevant substrates46. MS-based in vitro kinase assays became a powerful tool, nevertheless the total number of human kinases employed thus far in the individual study is still quite limited (less than 15).Recently, peptide arrays using a synthetic peptide library with randomized sequencesin vitro kinase assay to profile three human protein kinases, Erk1, AKT1 and PKA, in which dephosphorylated lysate proteins were used as the substrate source for in vitro kinase reaction and the phosphorylated proteins were digested by trypsin, fractionated by ion-exchange StageTip, enriched by titania chromatography and analyzed by LC-MS/MS47. Thousands of in vitro phosphorylation sites for each kinase were successfully identified and several new phosphorylation motifs together with the known motifs reflecting the specificity of each kinase were found. In this study, we modified the previous approach for kinome-wide applications, in which a stable isotope-based quantitation approach was used through the study to reject the endogenously phosphorylated proteins remaining in the substrate source even after phosphatase-treatment. By using this approach, 385 human recombinant kinases consisting of 354 wild-type protein kinases, 21 mutants and 10 lipid kinases were analyzed to acquire an in vitro substrate library as well as phosphorylation motifs. Substrate preference of each kinase was more accurately obtained by using quantitative phosphoproteomic approach with an isotopical labeling to reject the contaminant phosphoproteins constantly through this study. Using these results, a novel ‘kinome tree’ based on kinase-substrate relationships was proposed.Previously, we developed an LC-MS/MS-based in vitro kinase assay .Based on the 52. The casein kinase 1 (CK1) and 2 (CK2) families and some other kinases showed phosphorylation of peptides containing D/E-rich motifs.Typical examples of extracted phosphorylation motifs are shown in Fig. Some protein kinases showed ‘unreported’ phosphorylation motifs that are different from already-known motifs Fig. , for exain vitro substrates of 21 tumor-associated mutant protein kinases and 10 lipid kinases (LKs) were also investigated in the same manner as described for the wild-type kinases. Tested LKs phosphorylated few proteinous substrates, and no significant phosphorylation motifs could be extracted . Although some common structures, such as R-X-X-pS or pS/T-P, were extracted as major phosphorylation motifs for many STKs in the motif analysis, the kinases were subdivided into small groups like families or subfamilies by the cluster analysis. This result indicates that the representative phosphorylation motif for each kinase is insufficient to describe kinase profiles precisely, and substrate-based classification has more power to distinguish subtle differences between kinases, even though the substrates were acquired under in vitro conditions. Conventional kinase classification  is based on sequence and domain similarity and cellular function of kinases2. The substrate-based classification pattern in our study was, in general, similar to the conventional kinome tree, especially in the CMGC, AGC, CAMK and CK1 groups. However, some clear differences were observed between the classifications, such as the difference in phosphorylation preference for serine, threonine and tyrosine. STKs with tyrosine preference are marked with # in Fig. 55. Recently, it was reported that Haspin and Camkk2b showed an in vitro Thr preference with synthetic and HeLa tryptic peptides43. In our in vitro experiments, most STKs were strongly directed to Ser as expected, but MAPK, CDK and some kinases had a relatively low Ser preference. Furthermore, most of the STE group and a portion of the kinases belonging to the TKL and Others groups preferentially phosphorylated Thr  at the DFG + 1 position, whereas Thr-directed kinases have branched aliphatic residues . They also reported that kinases without any Ser/Thr preference have Leu or Ser at the DFG + 1. Our results based on 255 human STKs also supported their finding that all of the kinases with Phe at the DFG + 1 position showed Ser preference  method57, in which phosphopeptides with different localization of phospho-group can be accepted when the spectrum contains different series of site-determining ions for each phosphosite.Since TSAP dephosphorylation is not complete, it is important to quantify residual phosphopeptides relative to peptides phosphorylated by spiked kinases. By removing residual phosphopeptides based on the relative ratio, the peptide sequences could be used with high confidence for motif extraction. Nevertheless, the phosphorylation position within the peptide sequence should be independently considered because the fragmentation of phosphopeptides is not always sufficient for unambiguous determination of the phosphosite. In this study, we used two approaches, in vitro kinase assays, it was reported that Haspin and Bmpr2 preferentially phosphorylate the N- or C-terminal positions of synthetic and HeLa tryptic peptides43. However, no such preference was observed in our study, in which proteins extracted from cell lysates were used as the substrate source, suggesting that peptides are not suitable as a substrate source for the extraction of phosphorylation motifs in some cases. In addition, tryptic cleavage may disrupt motif sequences, especially for basophilic motifs. Therefore, we employed proteins as the substrate source, although they were heat-treated to inactivate TSAP.Regarding the differences between proteins and peptides as substrate sources for 58 as well as CAMKK259. The parent kinase LKB1 is grouped with CAMKK1, CAMKK2 and PBK in the in vitro assay, whereas LKB1 is not in the same group as CAMKK1, CAMKK2 and PBK in the conventional kinome tree. This indicates that the kinase classification based on the in vitro assay data is more reliable to predict the physiological substrates in some cases. Note that the substrates commonly phosphorylated by the classified 4 kinases mainly contain L-X-pT or pT-X-X-G, both of which are consistent with the T-loop structure of AMPK-related kinases. Mapping of our classification to the classical kinome physiological tree indicates that kinases belonging to CMGC group, CK1 group and both of AGC and CAMK groups are exclusively proline-directed, acidophilic and basophilic, respectively , however our dataset covered only approximately 4% of the kinase-substrate relationships published in PhosphoSitePlus, regardless of whether they were observed in vitro assay. This very large dataset of kinase-substrate pairs is expected to be a core resource in the development of more reliable computational tools to uncover the overall phosphorylation network in cells.In conclusion, we profiled substrate preference of the 385 human kinases using 2H2, 13C]Formaldehyde was purchased from ISOTEC . Recombinant human protein kinases were from Carna Biosciences , with the exception of KHS1, SGK496, NIK, CDK6/Cyclin D1, CDK9/Cyclin K, ALK1, CDK4/Cyclin D1, BARK1 and MPSK1  and GPRK5, FRAP, DAPK2, PKG1a, STK33, TAO1 and ULT3 . More details on kinases are shown in Supplementary Table Titanium dioxide (titania) particle (10 μm diameter) were purchased from GL Sciences . Thermo-sensitive alkaline phosphatase (TSAP) and modified trypsin were obtained from Promega . - or  dimethylation of amino groups at the peptide N-terminus and in the lysine side chain were allowed as variable modifications.Database searching was performed as described70. The peak area of identified phosphopeptide was integrated using Mass Navigator v1.2, and then the peak area ratio for kinase-treated to control sample was calculated. Peptides for which the peak area ratio was less than or equal to 2 and incompletely modified peptides were rejected. Decoy database search using a randomized database created by a Mascot Perl script gave a 0–0.3% false-positive rate for identified peptides with these criteria. Phosphosite localization was evaluated by using an in-house Perl script to check for the presence of a site-determining ion combination57, and PTM scores were obtained with PhosCalc version 1.271.Peptides were considered identified if the Mascot score was over the 95% confidence limit based on the ‘identity’ score of each peptide and if at least three successive y- or b-ions with a further two or more y-, b- and/or precursor-origin neutral loss ions were observed, based on the error-tolerant peptide sequence tag concepthttp://motif-x.med.harvard.edu/)51, in which the minimum number of motif occurrences was set to 10, the significance threshold was set to 10e-6 and the IPI Human Proteome Database was used as a background.Using the amino acid sequences of phosphopeptides that were increased more than 2-fold with kinase reactions, all proteins containing each peptide sequence were extracted from the SwissProt database. Then, phosphoacceptor-centered 13-amino-acid sequences were extracted from the matched protein sequences, and redundant sequences were removed. Phosphorylation motifs of each kinase were extracted using Motif-x , and 2 units of TSAP was added. After incubation at 37 °C for 60 min, the solution was desalted with a SDB-XC stage tip and evaporated in a vacuum concentrator. The residue was taken up in 10 μL of solution A for subsequent nanoLC-MSMS analysis.The kinase reaction mixture was divided into 2 aliquots, which were labeled with normal or Supplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5


These three characteristic peaks are indicators of the phase transition between ice VII and VIII and ice X. Despite many experimental and theoretical works on ice X, only this study has clearly indicated these characteristic peaks in the region of the IR band. The phonon density of states shows quite different features than ice VIII, which could be verified by inelastic neutron scattering in the future. The dynamic processes of 15 vibrational normal modes are discussed and the typical hydrogen bonds are missing.A typical vibrational spectrum in the ice phase has four separate bands: Translation, libration, bending, and stretching. Ice X, the final ice phase under high pressure, shows an exotic vibrational spectrum. Based on harmonic approximation, an ideal crystal of ice X has one peak, at 998 cm Water is one of the most abundant materials. It is important for both nature’s balance and human evolution. It is essential in both Earth and planetary sciences to understand the behavior of water over a broad range of thermodynamic conditions and experiments . As the −1 at pressure above 175 GPa as an indicator of ice X. Their calculations indicate that there are two IR absorption peaks at 1508 cm−1 and 450 cm−1 at pressure above 120 GPa.As early as 1972, Holzapfel was the first to predict a high-pressure phase of ice that exists with hydrogen-bonded protons residing in the middle of two symmetrically neighboring oxygen atoms . In 1984Pn3m structure at high pressure [−1 [−1 and 785 cm−1. However, a unique Raman peak for ice X has not been proposed. In this study, to clarify the critical indicators of the transition from ice VIII to X, 15 normal vibration modes of ice X are individually analyzed and three vibrational spectra are illustrated for reference to the phase transition.Two symmetric O–H bonds connect each hydrogen atom in the ice X crystal. Consequently, it is no longer in a molecular phase, but in an atomic one . Considepressure . In addisure [−1 . Alexandsure [−1  and Men sure [−1  observedI41/amd. As shown in Pn3m at 120 GPa. Then, the phonons and IR properties of ice X were calculated to simulate the phonon density of states (PDOS) and the photon scattering spectra at this pressure. The normal vibration modes were extracted from the property calculation of the polarizability, IR, and Raman spectra. The exchange–correlation function of the generalized gradient approximation RPBE [−9 for the PDOS calculation with norm-conserving pseudopotentials.Using CASTEP , a firstion RPBE  was sele−1. There were no frequencies at this region for ice X, meaning that strong covalent bonds were missing. The PDOS peaks of ice X below 300 cm−1 were from the acoustic branch and the band from 400 cm−1 to 1000 cm−1 was from the optic branch. Obviously, the intermolecular hydrogen bonds were also missing. These features at above 400 cm−1 were similar to the librational bands of other ice phases. However, the stubborn isolated intramolecular O–H bending peaks, at approximately 1600–1700 cm−1, extended to a broad band. On account of the high pressure, ice X demonstrated metallic properties to some extent. It is unsurprising that the spectrum of ice X was quite different from the spectra of traditional ice phases. The PDOS curve reflected phonons integrated by the entire reduced Brillouin zone. As the signals collected by the INS experiment were proportional to the PDOS, this simulated curve may be verified via INS in the future. In spite of its PDOS complexity, the IR and Raman spectra showed very distinct indicators of ice X identification. Our simulations showed that two peaks, at 450 cm−1 and 1508 cm−1, were IR active and that only one peak, at 998 cm−1, was Raman active . In the IR study of Goncharov et al., it was obvious to assign a peak at approximately 1500 cm−1 in the experimental spectrum [−1 [−1 and 785 cm−1, respectively. As vibrational frequencies are very sensitive to pressure, big differences in peak position may reflect various pressure conditions. For the first time, we theoretically confirm three indicator peaks in ice X, one for Raman scattering and the other two for IR absorption.trum [−1 . Howevertrum [−1  and Men trum [−1  observedThe periodic structure of ice X is a six-atom primitive cell, which greatly reduces the normal vibrational modes. Ice X has 3 × 6 − 3 = 15 optic modes. The 15 modes are discussed individually below.−1 [−1 . However−1 . We foun−1 had all oxygen atoms vibrating, whereas the hydrogens remained static in the lattice. As their relative vibrational directions were opposite, we labeled the directions of oscillation to distinguish these three modes, which were roughly depicted as , , and . There was little deviation from these three directions. We confirm that they were three orthorhombic vibrational directions and that they were sensitive to different pressures. This was the only Raman active peak based on harmonic approximation. In their work on Raman spectra, Putrino and Parrinello [−1. Their experimental data indicated that the peak occured at 840 cm−1, which agrees with our result at 998 cm−1. The reported anharmonic motions cannot be calculated in this work. −1.The three degenerate states at 998 cmrrinello  reported−1. These modes were similar to the twisting modes in the librational bands of other ice phases. As shown in There were three rotating modes at 1260 cm−1 in which the four O–H–O bonds bent in the same direction (three were strong and one was weak). These three degenerated modes composed the other peak of IR active. The three directions of degenerate modes at 1508(1), 1508(2), and 1508(3) were approximately equal to , , and , respectively. −1 and one isolated mode at 2623 cm−1. In the case of 2115(1), the two hydrogen atoms oscillated in the opposite direction along , whereas the other two moved along . For 2115(2), the mode could be regarded as one H–O–H bending in the  direction, whereas the other one vibrated in the  direction. Vibrational frequency at 2623 cm−1 was a unique mode, which was the upmost frequency. In this mode, the four hydrogen atoms simultaneously vibrated toward the central oxygen. This was a symmetrical motion through the center of inversion and was, of course, IR and Raman inactive. Interestingly, there were two degenerate modes at 2115 cm−1, for Raman scattering and two peaks, at 450 cm−1 and 1508 cm−1, for IR absorption. These three peaks are indicators of complete ice X phase transition. Phase transition was observed above 60 GPa. We conclude that the transition region ranges from 60 GPa to 120 GPa. The complex vibrational peaks observed by experiments were from incomplete phase transition in this range. As the vibration frequencies are pressure-dependent, the pressure conditions should be examined when considering the three peaks mentioned above.Fifteen normal vibrational modes of ice X are discussed based on the DFT calculation. The PDOS integrated by dispersion curves showed quite complex features. However, photon scattering theoretically presented very simple features—one peak, at 998 cm


The earliest Neolithic of southwest Asia is generally perceived and portrayed as a period of emerging economic practices that anticipated full-fledged food-producing economies. This first Neolithic, however, can also be seen as the last gasp of an earlier way of life that remained fundamentally Epipaleolithic in character. While people at this time had begun to cultivate some of the plant foods gathered in preceding periods, and to live for lengthy periods in sites with substantial architecture, they also relied on hunting for a significant portion of their diet and logistical movement across landscapes to exploit diverse environments. The objective of our research on Nachcharini Cave, the only excavated early Neolithic site in the high mountains of northeastern Lebanon, is to evaluate its role in a form of logistical organization not well attested at other sites in the Levant during this period. On the basis of material that Bruce Schroeder excavated in the 1970s, we present here for the first time analyses of faunal and lithic evidence from Nachcharini Cave, along with new radiocarbon dates that place the major occupation layer of the site firmly in the earliest Neolithic. We conclude that Nachcharini was a short-term hunting camp that was periodically used over some two centuries. The prevailing picture of the shift from mobile hunter-gatherer adaptations to village farming communities in the Middle East portrays a complex process in which changes in settlement, ritual, and mobility preceded clear morphological evidence for subsistence based on domesticated crops and subsequently domesticated animals –4. In thWe present here the first comprehensive overview of the results of Bruce Schroeder’s excavations at the PPNA site of Mugharat an-Nachcharini in the Anti-Lebanon Mountains , includi‘Ard al-Kichek plateau of the Anti-Lebanon Mountains  in eastern Lebanon, near the modern border with Syria [Nachcharini Cave  is locatth Syria –14. Unli2 and pointed to the richness of the PPNA component . These were corrected for fractionation by using measured The faunal material from Nachcharini Cave (layer 4d) is generally well preserved in terms of surface details and overall condition but the specimens are highly fragmented, primarily as a result of intentional perimortem fracture, presumably for marrow extraction. Various post-depositional factors also contributed to fragmentation in some cases. Therefore, the vast majority of specimens cannot be attributed to a specific taxon. Despite these limitations, a significant number of specimens are identifiable to genus or species, providing valuable information about human activities at the site. Based on those ungulate faunal specimens with secure taxonomic attributions, this preliminary faunal analysis reports the Number of Identified Specimens (NISP) for the assemblage, and the Minimum Number of Individuals (MNI) for each taxonomic group. The MNI presented here is based on specimens of right mandibles, which were the most frequently occurring element type in the sample assemblage from Layer 4d. Identification is based on comparisons with the Howard Savage Archaeo-Osteology Collection, University of Toronto, and the Mammalogy Collection, Royal Ontario Museum, as well as established reference material –25 AdditLooking first at calibrations without assuming any model for their order or association (an “uninformative” model), OxA-28000 intercepts a plateau in the calibration curve, resulting in a multimodal calibration and relatively long time-span, while OxA-27999 intercepts a steep portion and OxA-27998 falls near the beginning of that portion. OxA-27998 does not overlap very much with the later two, even at 95% confidence, and may pertain to an earlier event see . In thisGrouping the later two determinations and comparing them with the first, with no assumptions about their relationship, widens the gap somewhat , likely showing sensitivity to the model assumption. Assuming that OxA-27999 and OxA-28000 pertain to the same (pooled) event, and OxA-27998 a different one predictably results in an even longer estimate of elapsed time between the older event and the younger one, 48 to 172 years at 68%. However, a test of the hypothesis that the older date falls within the interval of the two younger dates yields a probability of 0.908, despite the fact that the model assumptions have exaggerated the difference between the older date and the other two.Whether the dates and their associated faunal remains pertain to a single event around 9200 cal BC or to a series of events over a century or two, they generally fall well within the range of PPNA sites with which they share some other aspects of material culture. At Netiv Hagdud, for example, and using the same methods as for the Nachcharini dates, the beginning of occupation appears to be ca. 9480–9155 cal BC and its end around 9255–8800 cal BC  . A probaThe collection in Toronto does not include the complete excavated lithic assemblage, consisting almost exclusively of formal retouched tools. We cannot be certain that all formal tools recovered are present, although the collection matches well with the counts of retouched tools found in the excavation records. Data in the excavation records make it clear that unretouched debitage and cores did not occur in the high densities that one would expect if a complete production sequence took place on site . The lowThe dominant tool types in the Nachcharini assemblage from layer 4d are Khiam points and Hagdud truncations  and 7. HThe association of Khiam points and Hagdud truncations at Nachcharini supports the argument that these pieces were elements in composite hunting tools –38. ImpaRegionally, there is high degree of inter-site variability in the ratio of points to Hagdud truncations. Although sample size is an issue, Nachcharini and Netiv Hagdud nonetheless appear to be the only sites with a balance between the number of points and Hagdud truncations see . While tOvis cf. orientalis) are the most represented taxon with at least seven individuals present, while Mountain gazelle (G. gazella) are represented by a minimum of five individuals. The undetermined Ovis/Capra category also has an MNI of five, and of these, four are identified as “probable Ovis,” and one as “probable Capra.” Capra  itself has an MNI of two. So, sheep and goat together represent a minimum of 14 individual animals, while gazelle represent five, and deer  just two. Again, MNI for Ovis, Capra, and Gazella here is calculated on the basis of right mandibles. Age information shows that the primary focus of hunting at PPNA Nachcharini was on adults of prime age, although several sub-adult animals are also present in the Layer 4d sample. Ovis cf. orientalis) from Nachcharini Cave layer 4d.Some differences in post-cranial element representation were apparent. The sheep/goat and gazelle bones show fairly complete representation of all skeletal regions , suggesting that primary butchery of these animals occurred within the cave. The deer bones show a different pattern, with a more uneven representation of skeletal regions and more highly fragmented specimens, perhaps suggesting offsite butchery or accumulation by scavengers. Again, these conclusions are tentative due to small sample size and various taphonomic factors, mentioned above. A comprehensive report on the taphonomy and fauna from all excavated units at the site is forthcoming .The fauna from Nachcharini layer 4d shows a distinct focus on ungulates, with little evidence of the “broad spectrum” of animal resources seen at many contemporaneous Levantine sites  . Within The faunal and lithic data from Schroeder’s excavation at Nachcharini Cave present a vivid picture of a task-specific site focused on the hunting of ungulates, especially sheep. The radiocarbon dates firmly place this occupation within the PPNA, nearly synchronous with the occupation of Netiv Hagdud in the Jordan Valley. Lithic data, including the ratio of Khiam points to Hagdud truncations, also indicates a strong typological similarity between these two sites in these two tool types and probably their use in projectile armatures. However, from a broader perspective, Netiv Hagdud’s lithic assemblage exhibits a diversity not found at Nachcharini. The Khiam points from Nachcharini have a high degree of expediency that is consistent with retooling and there is no evidence for primary core reduction at the site. It appears that the PPNA hunters at Nachcharini were adept at using available materials to retool their hunting projectiles as needed. The dense accumulation of fauna in layer 4d at Nachcharini is consistent with intensive butchery, although it will require further taphonomic analysis to elucidate the nature of faunal exploitation. The three radiocarbon dates from the site are reasonably close but leave open the possibility that Stratum 4d is a palimpsest of multiple site visits over a period of up to two centuries.Nachcharini provides clear evidence that PPNA mobility included task-specific groups that moved seasonally to exploit resources well outside the vicinity of large sites with architecture. The fauna from Nachcharini layer 4d shows a distinct focus on ungulates, with little evidence of exploitation of the “broad spectrum” of animal resources seen at many contemporaneous Levantine sites, and a strong predominance of caprinae over other ungulates. Furthermore, these unusually exhibit more sheep than goat, affording the possibility of incipient herd management at this early date –45. NachThe reanalysis of material from Schroeder’s excavation at Nachcharini demonstrates not only the importance of this site but also some of the limitations of our understanding of PPNA society. The focus of research on large village sites, which, on the basis of evidence from Nachcharini, can be understood as base camps within a residential mobility strategy, probably provides only a partial perspective. Sites such as Iraq ad-Dubb, which is perched near the top of a cliff with limited space for occupation yet still has architectural remains dating to the PPNA , likely The PPNA was a dynamic period with diverse adaptations that appear to have included plant cultivation and perhaps incipient herd management. This period also saw radical developments in site structure and monumental architecture in some locations. PPNA groups voyaged widely, as indicated by expansion into Cyprus –58 and rS1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


ICK  gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.Loss-of-function mutations in the human  Intestinal cell kinase (ICK) is a highly conserved serine/threonine protein kinase in the CMGC (CDK/MAPK/GSK3/CLK) group of the human kinome ,2. ICK wICK gene have been identified in ciliopathies SKGKARPKTGC at GenScript . Phosphopeptide-specific antibodies were affinity-purified through a positive selection over phosphopeptide antigens followed by negative selections over non-phosphopeptide antigens. GST-tag (B-14) mouse monoclonal (sc-138) and HA-tag (12CA5) mouse monoclonal (sc-57592) antibodies were from Santa Cruz Biotechnology . KIF3A (D7G3) rabbit monoclonal (#8507) and HA-tag (C29F4) rabbit monoclonal (#3724) antibodies were from Cell Signaling Technology . Arl13B rabbit polyclonal antibody (17711-1-AP) was from Proteintech . Goat anti-rabbit IgG (Alexa Fluor 594) preadsorbed antibody (ab150084) was from Abcam . 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/L glucose and 10% fetal bovine serum (FBS) or 10% new born calf serum (NBCS). HEK293T cells were transfected using a calcium phosphate protocol as described in -ATP (PerkinElmer) in kinase buffer  at 30 °C for 5–30 min. The reaction was quenched by the addition of 2× SDS sample buffer and the samples were processed and analyzed for radioactivity by autoradiograph as described in [For in vitro analysis of KIF3A phosphorylation by CAPK, HA-KIF3A was expressed in HEK293T cells, affinity-purified from cell extracts by anti-HA Sepharose beads. HA-KIF3A substrates (0.1–0.5 μg) were incubated with active His-CAPK1–291 ,22 proteribed in ,22. For For in vivo analysis of KIF3A phosphorylation by CAPK, HA-KIF3A and GST-CAPK were co-expressed in HEK293T cells. HA-KIF3A proteins were immunoprecipitated from cell lysate and captured on anti-HA Sepharose beads. The reactivity of HA-KIF3A against the KIF3A-phospho-Thr672 antibody was analyzed by Western blot.GST-CAPK was expressed in HEK293T cells and affinity-purified by glutathione Sepharose beads. GST-CAPK beads were incubated with cell lysate containing HA-KIF3A proteins at 4 °C overnight, rinsed extensively, and eluted with 2× SDS sample buffer at 95 °C for 5 min. Eluate was Western blotted against HA or KIF3A antibody for detection of HA-KIF3A proteins bound to GST-CAPK. NIH-3T3 cells grown on collagen-coated coverslips were fixed by 4% paraformaldehyde (PFA) in PBS, rinsed in PBS, and then permeabilized by 0.2% Triton X-100 in PBS. After one hour in blocking buffer , NIH-3T3 cells on cover slips were incubated with cilia marker Arl13B rabbit antibody at 4 °C overnight followed by rinses in PBS and one hour incubation with anti-rabbit IgG (Alexa Fluor 594-conjugated) secondary antibody. After extensive rinses, slides were mounted in antifade reagent containing DAPI  for imaging . t-test. Data were reported as mean ± standard deviation (SD). P-values less than 0.05 were considered as significant.Quantified experimental data were analyzed by the student 32P radioisotope labeling and a phosphosite specific KIF3A-phospho-Thr672 antibody, to show that KIF3A-Thr672 is phosphorylated by CAPK in vitro  carboxyl-terminal domain (CTD) that shares no significant similarity with known proteins A. We speFurthermore, we conducted an in vitro binding assay to evaluate the interaction between CAPK and KIF3A C. This aNext, we asked the question if the CTD of CAPK influences phosphorylation of KIF3A in vivo. We co-expressed GST-CAPK WT (wild type), KD (kinase dead), or CTD-truncation (1–291) with HA-KIF3A in HEK293T cells. We assessed KIF3A-phospho-Thr672 signals in whole cell extracts and anti-HA immunoprecipitates by Western blotting . Our resMounting evidence has shown localization of CAPK at the primary cilium in cells ,15,16,23Since the CTD of CAPK is required for ciliary targeting and binding, and phosphorylation of its ciliary substrate KIF3A, we hypothesized that the CTD is essential for CAPK function in suppressing the formation of primary cilia. We examined ciliogenesis by transfecting NIH-3T3 cells with plasmids encoding GFP-CAPK WT, R272A, or CTD-truncated (1–291) and quantifying ciliated cells in GFP-positive versus GFP-negative cells (as controls) using a ciliary marker, Arl13B. Expression of GFP-CAPK WT reduced the number of ciliated cells by 25%, whereas neither the loss-of-function R272A mutant CAPK protein or the CTD-truncated mutant, GFP-CAPK (1–291) had a significant effect on ciliogenesis . Cells ehttp://distilldeep.ucd.ie/porter/) [Although CAPK is very similar to classic MAPKs in the N-terminal catalytic domain, it diverges significantly in both the length and sequence of the C-terminal non-catalytic domain (CTD). No structural domain or motif has been found in this CTD (285–632 aa) using NCBI’s conserved domain architecture retrieval tool (CDART) . Furtherporter/) . This suporter/) . Even thporter/) . We prevA lack of the CTD compromises both ciliary localization and the ability of CAPK to bind and phosphorylate KIF3A. This raises the question of whether cilia localization or phosphorylation of KIF3A is important for the CTD-dependent CAPK effect on ciliogenesis. By comparison R272A is a kinase inactivating mutation [We do provide new compelling evidence supporting human KIF3A-Thr672 (mouse Kif3a-Thr674) as an in vivo phosphorylation site of CAPK. This CAPK site is in the C-terminal region of human KIF3A that contains multiple phosphorylation sites, including PKA site Ser687 and CaMKII sites Thr692 and Ser696 . PhosphoCAPK is a highly conserved modulator of primary cilia structure and function. From this study, we conclude that both the catalytic activity and the intrinsically disordered non-catalytic C-terminal domain (CTD) of CAPK are required for the suppression of ciliogenesis. Here, we propose two mechanisms by which the CTD of CAPK is involved in its ciliary function. Firstly, CAPK localization to the primary cilium depends on the presence of the CTD. Secondly, the CTD facilitates CAPK interaction with and phosphorylation of its ciliary substrate KIF3A.