extfieldsofstudy: Biology Medicine

text:
Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO.

Introduction
L-Amino acid oxidase (LAAO; EC 1.4.3.2) is usually a flavin adenine dinucleotide (FAD)containing homodimeric protein, which functions in stereospecific oxidative deamination of L-amino acids to the corresponding a-keto acids with release of NH 4 + and H 2 O 2 [1]. It plays important biological roles, such as apoptosis [2], cytotoxicity [3], edema [4], hemolysis, hemorrhage [5], inducing or inhibiting platelet aggregation [6], and parasite-killing and antimicrobial activities [7]. Most of its biological functions are associated with the produced H 2 O 2 which is toxic to most organisms [8,9]. Therefore, LAAO is considered as a toxic protein and characterization of the mechanisms controlling its expression is very important for understanding not only its biological but also ecological roles. The enzymatic and physiochemical properties, biological function and structure of LAAO as well as its cloning and heterologous expression have been intensively investigated [9]. However, studies on unraveling the mechanism regulating LAAO expression are limited, where only a few genes controlling its transcription and translation have been explored. Previously, it was found that LAAO activity in Neurospora crassa can be induced by L-Phe, D-Phe, ATP and cycloheximide. Those inducing agents can regulate the lao gene expression at transcriptional level [10]. However, the regulation genes were undefined. More recently, a spontaneous mutant of N. crassa gln-1br8 with deficiency in glutamine synthetase β polypeptide was confirmed to have higher LAAO activity [11]. Again, the relevant genes for regulation were unexplored. It was also reported that LAAO activity in N. crassa can be induced through addition of L-amino acids to nitrogen-starved cultures as well as addition of protein synthesis inhibitors or Damino acids [12,13]. LAAO expression was regulated by NIT2 and the nmr gene product at transcriptional level [14]. Besides LAAO in N. crassa, LAAO from Marinomonas mediterranea was found to be regulated by hybrid sensor histidine kinase PpoS at transcriptional level [15]. Two genes of lodAB operon were found to be necessary for LAAO expression in M. mediterranea [16].
LAAO is widely found in marine Pseudoalteromonas spp. [17][18][19] and plays an important role in dispersal and colonization across a range of Gram-negative marine bacteria, such as Pseudoalteromonas tunicate [20], suggesting its important ecological function in marine environment. Recently, a yellow-pigmented marine bacterium Pseudoalteromonas sp. B-3 was isolated to produce LAAO [21,22]. In this study, to explore the genes involved in regulating LAAO activity, a red-pigmented spontaneous mutant strain of Pseudoalteromonas sp. B-3 resistant to rifampicin, designated as Pseudoalteromonas sp. Rf-1, was selected. The transposon mutagenesis [23] was used to construct a mutant library of strain Rf-1 and the target mutants with altered LAAO activity were screened using Prussian blue agar assay method [22]. Based on genetic and enzymatic analysis, several regulation genes were identified. Our results shed some lights into the molecular mechanisms regulating LAAO activity in this marine bacterium across the natural environment.

Mutant screening of LAAO activity
To explore the genes regulating LAAO activity in Pseudoalteromonas sp. Rf-1, plasmid pLOF/Km carrying a mini-Tn10 [24] was used to generate a mutant library of strain Rf-1. Antibiotics of rifampicin (Rif) and kanamycin (Km) were used to repress Escherichia coli with plasmid pLOF/Km and wild type strain Rf-1, respectively. After transposon mutagenesis [23,25], approximately 4,000 mutants appeared on marine medium (MM) plate with Rif and Km. Then, culture supernatant of individual mutant was subjected to LAAO-activity screening with Prussian blue agar assay [22]. Altered LAAO activity was detected in 300 mutants,(data not shown) in which 15 mutants displayed significant altered or abolished LAAO activity (Fig. 1). As shown in Table 1, compared to wild-type strain, 3 mutants (B3, B21 and B22) gave increased LAAO activity, 8 mutants (B9, A15, A45, B20, B17, A60, B10 and B11) yielded decreased LAAO activities, while the other 4 mutants (B19, B12, B6 and B1) showed null LAAO activity. The statistical analysis showed that the mean difference of blue halo diameter or LAAO activity between mutant and wild type was all extremely significant (P<0.001) ( Table 1). Therefore, these 15 mutants were selected for further investigation. Measurement of LAAO activity from the LAAO-altered or -deficient mutants using Prussian blue agar assay [22]. 50 μl of culture supernatant of individual mutant or wild-type strain Rf-1 was added into the punched circular hole with a diameter of 6 mm in the Prussian blue agar assay plate for color development from Berlin green to blue caused by H 2 O 2 produced by LAAO activity. The diameter of the resultant blue halo is exponentially associated with the LAAO activity. Compared with wild-type strain, the mutants B3, B21 and B22 had the increased LAAO activities, and the mutants B9, A15, A45, B20, B17, A60, B10 and B11 yielded the decreased LAAO activities, while the mutants B19, B12, B6 and B1 were deficient in LAAO activity.

Molecular confirmation of transposon insertion in mutants
Two primer pairs were used to confirm the correct insertion of mini-Tn10 gene along with Km resistance marker gene (IS-km-IS) in the 15 mutants from above. One pair was Tn10KAP3 (5'-CATTTGATGCTCGATGAGTTTTTCT-3') and S1 (5'-TTGGTAAAAATCATTAAGT-TAAGGT-3'), which were both specific to IS-km-IS region; the other pair was DTn10AP3 (5'-CGTTGCGCTGCCCGGATTACAGCCG-3') and Q2 (5'-AAACGATGCCCATTTTGTT-GATTAT-3'), which were specific to IS-km-IS and pLOF vector region (outside of IS-km-IS), respectively. The first primer pair was used to amplify a 506bp fragment, while the second one was use to amplify a 964bp fragment. As shown in Fig. 2, positive control of pLOF/Km yielded both fragments, while negative control (wild type strain Rf-1) gave neither. Mutants A60 and A15 exhibited neither fragment, suggesting unclear mutation or failure of PCR amplification; mutant A45 unexpectedly displayed both fragments, probably due to insertion of extra piece from pLOF/Km vector or even whole pLOF/Km. On the other hand, all the remaining 12 mutants expectedly gave only one fragment (506 bp) from primer pair of Tn10KAP3 and S1, but not the other one (964 bp) from primer pair of DTn10AP3 and Q2. All these results indicated that the correct insertion of IS-km-IS took place probably only in 12 mutants (B3, B21, B22, B9, B10, B11, B17, B20, B1, B6, B12 and B19) which were used in the following experiments. PCR detection of insertion of mini-Tn10 together with kanamycin resistance marker (IS-km-IS region) in LAAO-altered mutants. S: PCR using primers Tn10KAP3 and S1; Q: PCR using primers DTn10AP3 and Q2; pLOF: PCR using plasmid pLOF/Km as template (positive control); Rf-1: PCR using genomic DNA of wild type strain Rf-1 as template (negative control); B3, B21, B1, B12, B19, A45, B6, B9, B10, B11, B20, A60 and A15: PCR reaction using genomic DNA from the corresponding mutant as template. Quantitative real-time PCR (qRT-PCR) analysis of the differential expression of lao gene The expression of lao gene in 12 mutants identified above and wild-type strain was measured by qRT-PCR [26]. The results in Fig. 3 showed that all the strains yielded the expression of lao gene. Compared to wild-type strain Rf-1, 3 mutants (B3, B21, and B22) had increased expression of lao gene, while the other 9 gave decreased expression. Overall, the qRT-PCR results showed the same tendency as LAAO enzymatic activity in the different mutants. In mutants with null activity, the qRT-PCR gave also lower levels of mRNA, while in mutants with higher LAAO activity it is possible to see higher mRNA levels ( Fig. 1 and Table 1). Notably, the mutants (B1, B6, B12 and B19) with abolished LAAO activity still exhibited lao gene expression. Statistical analysis indicated that the mean difference of relative lao gene expression between mutant and wild type was significant (P<0.05) in mutants B19 and B17, and extremely significant (P<0.001) in mutants B1, B6, B12, B9, B10, B11, B20, B3 and B21, but not significant (P>0.05) in mutant B22. However, regarding LAAO activity, the mean differences between mutants and wild type all were extremely significant (P<0.001). Thus, all these findings indicated that LAAO activity and the corresponding lao gene expression are probably regulated by the disrupted genes in different mutants at different levels.

Characterization of the genes disrupted by transposon in mutants
The DNA region flanking inserted transposon was sequenced for identification of the disrupted gene in each mutant. Typically, a DNA sequence with around 500-1200 bp for each mutant was obtained through hiTAIL-PCR [27]. As shown in Table 2, most of the sequences in mutants could match with the genes from the same genus, Pseudoalteromonas rubra ATCC 29570, with the exception that mutant B10 showed the highest similarity (68%) to a gene coding for TonB-dependent heme receptor family in Alcanivorax dieselolei B5. The disrupted gene resulting in increase of LAAO activity in mutant B22 showed the highest identity (72%) to a gene encoding GntR family transcriptional regulator. The possible corresponding genes resulting in decrease of LAAO activity in mutants B20, B17 and B10 had the closest similarity to the genes coding for methylase (80%), non-ribosomal peptide synthetase (82%) and TonBdependent heme receptor family (68%), respectively. Interestingly, the disrupted genes in mutants B1, B6, B12 and B19 with abolished LAAO activity had the highest similarity to those coding for SAM-dependent methytransferase (80%), Ketol-acid reductoisomerase (80%), Nacetyltransferase GCN5 (80%), and Na + /H + antiporter NhaD and related arsenite permease (81%), respectively. Besides, the disrupted genes in mutants B3 and B9 yielded no any similar gene in databases and the ones in mutants B21 and B11 all matched with the unannotated sequences. Therefore, we did not try to propose the function of these 4 genes. All the characterized gene sequences have been deposited in GenBank and the corresponding access no. was included in Table 2.

Complementation of the disrupted genes in mutants with abolished LAAO-activity
The transposon-disrupted genes in the 4 mutants with null LAAO-activity (B1, B6, B12 and B19) were identified as sdmT, karI, nat5 and, nhaD with their corresponding functions displayed in Table 2. To further confirm that these identified genes were involved in regulation of LAAO biosynthesis, we tried to perform gene complementation in each mutant. We retrieved the full gene sequences of sdmT (access no. KP191657), karI (access no. KP191658) and nhaD (access no. KP191659), with length of 594 bp, 1473 bp and 1455 bp, respectively. However, the retrieval of full nat5 gene failed, even after extensive optimization procedure on hiTAIL-PCR condition. Subsequently, the 3 successfully retrieved genes were cloned into vector pBBR1MCS-5 [28] to form pBBR1MCS-5/sdmT, pBBR1MCS-5/nhaD and pBBR1MCS-5/karI, respectively. The pBBR1MCS-5/sdmT and pBBR1MCS-5/nhaD were successfully transformed into mutants B1 and B19, respectively, with assistance of plasmid pBR2013 [29] in E. coli DH5α. However, the transformation of pBBR1MCS-5/karI into mutant B6 failed. Prussian blue agar assay in Fig. 4 showed that the two complemented strains (complons), B1/MS and B19/MN yielded the Prussian blue halos with comparable diameter to wild-type strain Rf-1, indicating that LAAO activity was nearly completely recovered. Accordingly, our data demonstrated that both sdmT gene and nhaD gene are involved in controlling LAAO biosynthesis in Pseudoalteromonas sp. Rf-1. As shown in Fig. 5, the relative expression level of lao gene in either mutant B1 or mutant B19 was extremely significantly (P<0.001) decreased, compared to that in wild-type Rf-1. The complons B1/MS and B19/MN resulted from the complementation of sdmT gene and nhaD gene in B1 and B19, respectively, exhibited extremely significant (P<0.001) increase of lao gene expression, as compared to original mutants. In addition, their lao gene expression levels were comparable to that in wild-type Rf-1 (no significance, P>0.05), suggesting that the complementation of transposon-inserted genes can almost completely restore lao gene expression. The negative control using complons B1/M and B19/M with blank complementation lack of gene of interest displayed similar expression level as original mutants (no significance, P>0.05). Taken together, we concluded that both sdmT gene and nhaD gene upregulate the lao gene expression at transcriptional level in Pseudoalteromonas sp. Rf-1. Moreover, considering that the disruption of sdmT gene and nhaD gene resulted in completely undetectable LAAO activity, but only partial decrease of lao gene expression, we proposed that both genes also regulated LAAO biosynthesis probably at posttranscriptional level in Pseudoalteromonas sp. Rf-1.

Discussion
Species of the genus Pseudoalteromonas, in particular color-pigmented ones, are generally found to be associated with marine eukaryotes and have antibacterial, algicidal, antifungal, or antiviral activity. Several Pseudoalteromonas isolates specifically deter the settlement of common marine fouling organisms. Thus, Pseudoalteromonas cells are advantageous in their contest for nutrients, space and colonization of surfaces, and are protected against predators grazing at surfaces [30,31]. LAAO is believed to be associated with Pseudoalteromonas' biological and ecological roles in marine environment [30,31]. Therefore, a thorough understanding of mechanisms controlling LAAO production is extremely important and is attracting a great number of interests.
Despite the prevalent application of mutagenesis technique, such as transposon mutagenesis, in mutant screening since the past decade, applying the detection method cheaply and efficiently for large-scale screening of mutants with different LAAO activities has proven challenging [32]. To date, very little is known about the regulation mechanism underlying LAAO transcription and production in marine bacteria. Previously, Prussian blue agar assay method has been developed with high efficiency and convenience, holding a promising application as a sensitive procedure for differentiating the mutants from a mutant library with altered LAAO activity [22]. In this study, over 4,000 mutants with Rif and Km resistance were isolated and screened using Prussian blue agar assay, where only about 300 (7.5%) mutants in total were found to have altered LAAO activity. Among them, more than 95% mutants only had slight changes in LAAO activity, resulting in the difficulty for clear differentiation of mutants, which in the future could be overcome by using ferric-xylenol orange formation (Fe Ⅲ -XO) agar assay method with higher sensitivity [32,33] for detecting LAAO activity. Nevertheless, the combination of mini-Tn10 mutagenesis with Prussian blue agar assay allowed us to successfully identify several genes potentially controlling LAAO production in Pseudoalteromonas sp. Rf-1. Those identified genes could be grouped into two categories, positive regulator and negative regulator, based on the change of LAAO activity in mutants listed in Table 2.
The only identified negative regulator is GntR family transcriptional regulator that was found in mutant B22. The GntR family is one of the most abundant and widely distributed groups of transcriptional regulators in bacteria [34]. It was reported that the gntR gene acts to repress transcription of itself as well as a series of genes immediately adjacent to it [35,36]. Mutation of gntR gene resulted in increased LAAO activity, indicating a downregulation role of gntR gene in Pseudoalteromonas sp. Rf-1 for LAAO biosynthesis. In addition, our data showed that its disruption only caused slight increase of lao gene expression. This could suggest that the gntR gene probably downregulates some genes at transcriptional level which in turn regulate LAAO production at posttranscriptional level. However, this postulation remains to be further determined.
On the other hand, we identified 7 possible genes that are likely involved in upregulation pathway of LAAO activity. Three of proposed genes coding for TonB-dependent heme receptor family protein (TdhR), non-ribosomal peptide synthetase (NrpS) and methylase led to decreased LAAO activity after mutation. TdhR is predicted to localize at bacterial membrane, and can sense signals from extracellular or intracellular messengers and transfer them across membrane, leading to transcriptional activation of target genes [37]. The disruption of this gene resulted in extremely significant (P<0.001) decrease of lao gene expression, thus suggesting that the tdhR gene in Pseudoalteromonas sp. Rf-1 may positively regulate LAAO biosynthesis at transcriptional level. Moreover, TdhR protein can interact with outer membrane receptor that can carry specific substrates into cytoplasm space [37]. Thus, it is possible that TdhR may also be involved in absorption of substrates for LAAO biosynthesis. The NrpS protein is generally responsible for introduction of amino acid, and can control the production level of the corresponding peptides. Besides, NrpS also has formyltransferase activity and hydroxymethytransferase activity. Therefore, the NrpS protein may affect the substrate absorption for LAAO biosynthesis and posttranscriptional modification of LAAO. Moreover, our data indicated that the disruption of nrpS gene extremely significantly (P<0.001) decreased lao gene expression, suggesting that nrpS gene in Pseudoalteromonas sp. Rf-1 probably participates in upregulation of LAAO biosynthesis at transcriptional level, too. Similarly, considering that the insertion of transposon into methylase gene in mutant B20 resulted in both extremely significant (P<0.001) decrease of lao gene expression and extremely significant (P<0.001) decrease of LAAO activity, we postulated that this gene is potentially involved in upregulation of LAAO biosynthesis at transcriptional level.
Finally, we identified 4 upregulating genes that would abolish LAAO activity if disrupted. The disrupted gene in mutant B19 probably codes for Na + /H + antiporter NhaD and related arsenite permease (NhaD). NhaD is a ubiquitous protein usually in cytoplasmic membrane and in membranes of many organelles, and plays a primary role in homeostatic mechanisms and transmembrane transport of substances, such as H 2 O 2 , protein and vitamins. It is proposed that the disruption of this gene may disturb LAAO secretion. In addition, the disruption of this gene caused significant decrease (P<0.05) of lao gene expression, thus suggesting that the nhaD gene in Pseudoalteromonas sp. Rf-1 may also indirectly upregulate LAAO biosynthesis at transcriptional level. The disrupted genes in mutants B12 and B1 matched with the ones encoding N-acetyltransferase GCN5 (NaT5) and SAM-dependent methytransferase (SdmT), respectively. These two proteins are responsible for acetylation of Lys and Cys residues, and methylation of Glu, His, Lys and Arg residues, respectively, both participating in posttranslational modification of proteins [38]. These amino acid residues account for a huge amount of amino acids in LAAO of Pseudoalteromonas sp. Rf-1 (data not shown). Thus, NaT5 and SdmT may play a role in posttranslational modification of LAAO. Besides, NaT5 is responsible for acetylating the wobble base of elongator tRNA Met by utilizing acetyl-coenzyme A (CoA) and ATP (or GTP) to form N 4 -acetylcytidine (ac4C) [39]. The ac4C formation at wobble base of elongator tRNA Met is thought to ensure the precise recognition of AUG codon by preventing misreading of near-cognate AUA codon [39,40], thus ensuring the correct initiation of protein translation of protein. SdmT can catalyze 2'-O-methylation of cytidine 1402 and N 4 -methylation of cytidine 1402 in 16S rRNA. It has been found that methylation modification in 16S rRNA is necessary for stringent selection of the initiator tRNA and efficient translation initiation at UUG and GUG [41,42]. All these suggest that both nat5 and sdmT genes in Pseudoalteromonas sp. Rf-1 may positively regulate the translation initiation of LAAO as well. Considering the fact that the disruption of these two genes extremely significantly (P<0.001) downregulated lao gene expression, it is clear that both genes are probably involved in positive regulation on LAAO biosynthesis also at transcriptional level. The disrupted gene in another mutant B6 with no LAAO-activity matched with the one coding for ketol-acid reductoisomerase (KarI). This enzyme can catalyze conversion of acetohydroxy acids into dihydroxy valerates, which is a synthetic pathway of the essential branched side chain of amine acids Val and Ile. Probably, the disruption of karI gene in Pseudoalteromonas sp. Rf-1 will affect the synthesis of amino acids Val and Ile in LAAO, thus leading to loss of LAAO activity. Since the karI gene disruption extremely significantly (P<0.001) downregulated lao gene expression, it is clear that the karI gene positively regulates LAAO biosynthesis also at transcriptional level.
To our best knowledge, it is the first time to explore many genes involved in regulation of LAAO activity in Pseudoalteromonas sp. Rf-1 at transcriptional, posttranscriptional, translational and/or posttranslational level. Although western blot and/or site-directed mutation will be desired to confirm the molecular function of the explored genes in LAAO biosynthesis, our findings pave a road for exploiting the mechanisms underlying LAAO biosynthesis and secretion, and provide the understanding of biological and ecological roles in Pseudoalteromonas sp. Rf-1 by controlling LAAO activity in marine environment.

Strains, plasmids and media
All bacterial strains and plasmids used in this study were shown in Table 3. Pseudoalteromonas sp. Rf-1 (Rif r ) was grown in a marine medium (MM) (0.3% yeast extract, 0.5% peptone, 3% sea salt (Sigma)) at 28°C with a shaking of 120 rpm. All E. coli cells were cultured in Luria-Bertani (LB) medium at 37°C with a shaking of 150 rpm. Unless otherwise specified, to make solid medium, agar was added to a final concentration of 2%. When required, antibiotics were added to medium with final concentrations as below: rifampicin (Rif) 50 μg/ml, ampicillin (Ap) 100 μg/ml, gentamicin (Gm) 50 μg/ml, streptromycin (Str) 50 μg/ml and kanamycin (Km) 50 μg/ml.

Conjugation and transposon mutagenesis
To generate mutants with altered LAAO activity, transposon mutagenesis [23,25] was performed in rifampicin-resistant Pseudoalteromonas sp. Rf-1. In brief, the strain Rf-1 was inoculated into MM with 50 μg/ml Rif and the E. coli S17-1(λpir) harboring suicide vector pLOF/Km inoculated into LB (0.5% NaCl) with 100 μg/ml Ap and 50 μg/ml Km. After overnight incubation, both strains were reinoculated into their own fresh medium without antibiotics and allowed to reach exponential growth phase. To transfer the mini-Tn10 transposon together with kanamycin resistance marker on pLOF/Km to strain Rf-1 cells through conjugation, a 40 μl sample of the exponentially growing recipient Rf-1 cells was spotted onto the surface of conjugation medium (LB-MM, obtained by mixing equal amounts of the two media) agar plate and allowed to dry before another 40 μl of E. coli donor cells were added to the top of previous spot. Controls with only strain Rf-1 or E. coli were also prepared. After overnight conjugation, the cells were then collected by scraping and suspended in 1 ml of MM. After appropriate dilution, the cells were plated onto selective MM with 50 μg/ml Rif and 50 μg/ml Km, and incubated at 28°C for 48 h. Finally, the target mutants with altered LAAO activity were screened using Prussian blue agar assay method [22].

Prussian blue agar assay for screening of mutants with altered LAAO activity
Mutants with deficient or altered LAAO activity were selected based on high throughput ingel determination method of Prussian blue agar assay [22]. In brief, individual mutant was grown in MM with Rif and Km for 2 d at 28°C with a shaking of 160 rpm. After measurement of absorbance reading (600 nm) and adjustment of cell density if necessary, 1 ml of the above culture was added to 250 ml conical flask containing 50 ml of MM with Rif and Km for fermentation at 28°C with shaking at 160 rpm. After 72-h fermentation, the culture supernatant was harvested with a centrifugation at 5 000 rcf (relative centrifugal force) for 10 min at 4°C. Then, the Prussian blue agar assay plate containing 1.0 g/l FeCl 3 Á6H 2 O, 1.0 g/l potassium Table 3. Bacterial strains and plasmids used in this study.

Determination of transposon-inserted gene
Genomic DNAs were extracted from the transposon-inserted mutants of strain Rf-1 using a bacterial genomic DNA extraction kit (GE, USA). The DNA sequence flanking the inserted transposon in a mutant was determined using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) [27] (first time). The primers used for hiTAIL-PCR were shown in Table 4. In the first round, PCR was performed in 20 μl of reaction mixture containing 2.0 μl of PCR buffer, 200 μM dNTPs, 1.0 μM of any one of the LAD primers [27], 50 nM DTn10AP1, Table 4. Primers used in high-efficiency thermal asymmetric interlaced PCR.  Table 5. The PCR products from the second or third round of PCR amplification were electrophoresed on a 1.5% agarose gel and the desired amplicons were purified using a gel extraction kit (Qiagen, CA, USA) for TA cloning with pMD19-T simple vector (TaKaRa, Dalian, China). After sequencing by Sangon Biotech (Shanghai, China), the intermediate sequences of disrupted genes in mutants were collected and compared with the relevant genes in the GenBank of NCBI, EMBI databases and/or Prosite. To retrieve the entire length of sdmT, karI and nhaD gene sequences from LAAO-deficient mutants B1, B6 and B19, respectively, the hiTAIL-PCR was employed again (second time) to amplify the 5'-and 3'-regions of sdmT, karI and nhaD genes. As shown in Table 2, specific primer sets were designed based on the intermediate region collected from the first time of hiTAIL-PCR. After hiTAIL-PCR (Table 3), the desired products were recovered from agarose with gel extraction kit for TA cloning. After sequencing, the contigs of 5'-region, intermediate region, and 3'-region were assembled for analysis.

Complementation of the disrupted genes through tri-parental mating
To complement the disrupted genes of sdmT, karI and nhaD in three mutants of B1, B6 and B19, respectively, the entire three genes of sdmT, karI and nhaD were individually amplified using three primer pairs (Table 6), SdmtF and SdmtR, KariF and KariR, and NhaDF and NhaDR, respectively. The Pfu DNA polymerase (Promega, USA) was used to amplify 594 bp of sdmT gene, 1473 bp of karI gene and 1455 bp of nhaD gene. After addition of 3'-adenine overhang by Taq DNA polymerase (TaKaRa, Dalian, China), the desired PCR product was gel- The Genes Regulating LAAO Expression in Pseudoalteromonas sp. Rf-1 purified and cloned into pMD19-T simple vector. After digestion with Hind III/EcoR I, Kpn I/ Hind III and Kpn I/Xba I, the sdmT-gene, karI-gene and nhaD-gene fragments were individually cloned into expression vector pBBR1MCS-5 [28] to yield recombinant plasmids pBBR1MCS-5/sdmT, pBBR1MCS-5/karI and pBBR1MCS-5/nhaD, respectively, and separately transformed into E. coli HB101. Then, with assistance of plasmid pBR2013 [29] in E. coli DH5α, these three constructed plasmids were introduced into mutants B1, B6 and B19, respectively, for complementation of the corresponding disrupted gene into each mutant cell through tri-parental conjugation which was performed as below. First, the mutant of strain Rf-1 (receptor), E. coli HB101 with corresponding recombinant plasmid (donor) and E. coli DH5α with pBR2013 (assistor) were separately inoculated into MM with 50 μg/ml Rif and 50 μg/mL Km, LB with 50 μg/ml Gm and LB with 50 μg/ml Km, respectively, and allowed to reach exponential growth phase. Then, 2 ml of each culture was centrifugated at 2 000 rcf for 5 min to collect the cells. After washing once, each cell pellet was resuspended in 0.5 ml of conjugation medium (mixture of 1/2 MM and 1/2 LB) and three resuspensions were mixed together for another centrifugation at 5 000 rcf for 5 min. After removal of supernatant, the cell mixture of mutant, HB101 and DH5α was resuspended in 0.2 ml of conjugation medium and dropped to sterilized microporous membrane (size diameter 1 cm; aperture 0.45 μm) on conjugation medium plate for overnight conjugation at 30°C. Finally, the microporous membrane was transferred to 3 ml of MM for washing with vortex. After appropriate dilution, 50 μl of cell solution was spread on MM with 50 μg/ml Rif, 50 μg/ml Km and 50 μg/ml Gm. After incubation at 30°C, the target colonies were confirmed with molecular analysis and used for detection of LAAO activity and lao gene expression.

Analysis of lao gene expression by qRT-PCR
To measure the lao gene (access no. KP165133 in GenBank) expression, qRT-PCR [26] was used to amplify cDNA products reversely transcribed from mRNA of mutants selected. In brief, individual mutant was grown in MM with Rif and Km for 2 d at 28°C with a shaking of 160 rpm. Next, after measurement of absorbance reading (600 nm) and adjustment of cell density if necessary, 1 ml of the above culture was added to 250 ml conical flask containing 50 ml of MM with Rif and Km for incubation at 28°C with shaking at 160 rpm. After 72-h cultivation, the culture supernatant was harvested with a centrifugation at 5 000 rcf for 5 min at 4°C. Then, the total RNA was extracted using an RNAiso Plus (Total RNA extraction reagent) kit (Sangon, Shanghai, China). RNA integrity was determined based on OD 260 nm /OD 280 nm ratio (>1.95), and 500 ng of DNA-free RNA with high-quality was reversely transcribed to cDNA in a 10 μl volume using PrimeScript RT Master Mix (Perfect Real Time) kit. After appropriate dilution, Table 6. Primers for the amplification of full length genes of sdmt, kari and nhad.

Primers
Oligonucleotide sequences Restriction sites b the cDNA was used for amplification of 108 bp lao gene fragment with primer set of laoF (5'-ATACGCCAAGTGCCTCAGTG-3') and laoR (5'-TCCGTCAGCCCGTTAAAGTC-3') by using the SYBR green (Tli RNaseH Plus) kit. The PCR was run on CFX Connect Real-Time System (Bio-Rad, Hercules, CA) with an amplification protocol consisting of an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/ elongation at 60°C for 30 s. Immediately after the final PCR cycle, a melting-curve analysis was made to determine the reaction specificity based on the observation of melting temperature from product. Unless otherwise specified, all the kits above were purchased from TaKaRa (Dalian, China). The cycle threshold (C T ) for each PCR was determined using STATVIEW software which automatically set the threshold signal at the log phase of the amplification curve. Several dilutions of each cDNA sample were assayed for gene of interest in order to obtain a linear regression between C T value (ranging from 10 to 30 cycles) and log of the cDNA. The amplification efficiency of gene was retrieved from the slope of that linear regression according to the formula E = 10 (−1/slope) . The 116 bp of housekeeping 16S rRNA gene fragment (access no. KP234252 in GenBank) was amplified using primer set of 16SF (5'-AAGAAGCACCGGCTAACTCC-3') and 16SR (5'-TCTCGCTTAATCAACCGCCT-3') and treated as the internal control to verify that there were equal amounts of target cDNA in all samples. The relative expression (RE) of target gene (TG) in mutant or complon compared to that in wild type Rf-1 (WT) was calculated by the comparative C T method [43]. In brief, the difference of C T between TG and reference 16S rRNA gene (RG) was calculated according to the formula: DC TðTGÞ ¼ C TðTGÞ À C TðRGÞ Then, the RE of TG compared to RG was obtained by the equation: Finally, the RE of TG in mutant or complon compared to that in WT was achieved as below: RE ¼ 2 ÀDC TðTG; Mutant or ComplonÞ =2 ÀDC TðTG; WTÞ For each experiment, triplicate reactions were performed. All Data are presented as mean ± standard error of the mean. Significance of mean difference of relative gene expression was statistically analyzed by ANOVA.
Supporting Information S1 Dataset. Expression level of 16S rRNA gene and lao in mutants and wild type strain Rf-1.


s2fieldsofstudy: Biology Chemistry

text:
Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1BS421A mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1BD239A mutant exhibited very low levels of p-S421 in cells but this was increased by KRASG12V. In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

Abstract The dual-specificity tyrosine-phosphorylationregulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B S421A mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B D239A mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS G12V . In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

Introduction
The dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an evolutionarily conserved family of protein kinases that are found within the CMGC group (CDK, MAPK, GSK and CLK families) of the eukaryote kinome [1][2][3][4]. The mammalian DYRKs are divided into class I (DYRK1A and DYRK1B) and class II (DYRK2, DYRK3 and DYRK4) based on sequence homologies. The DYRKs require phosphorylation of the second tyrosine in the characteristic tyrosine-X-tyrosine (Y-X-Y) motif within their activation loop for kinase activity [5,6]. In contrast to many other kinases, this activation loop phosphorylation is not catalysed by an upstream, 'activating kinase' but occurs during translation when DYRKs undergo intramolecular cis-autophosphorylation on tyrosine [7]. As co-translational activation loop tyrosine phosphorylation is a one-time-only event, it was proposed that once synthesised, the mature DYRKs lose tyrosine kinase activity and phosphorylate substrates exclusively on serine or threonine residues. However, recent studies have demonstrated that mature DYRK1A retains the ability to autophosphorylate on other tyrosine residues outside the activation loop [8]. Regardless, substrate phosphorylation appears to occur exclusively on serine or threonine, usually within a proline-directed context (pS-P or pT-P) [9,10]. The DYRKs have roles in controlling transcription, mRNA splicing, cell cycle progression, survival and differentiation [2,3]. For example, loss of a single copy of Dyrk1a in mice leads to increased apoptosis and decreased brain size in mice [11]. In addition, the DYRK1A gene is situated in the Down's syndrome (DS) critical region on chromosome 21 (21q22. 1-22.3), is over-expressed in both foetal and adult brains of DS individuals [12] and is thought to contribute to the clinical features of Down's syndrome [13][14][15]. Indeed, Dyrk1a is the triplicated gene that causes decreased nuclear Cyclin D1 levels and early cortical neurogenic defects in a mouse model of DS [16]. Together, these studies emphasise the importance of DYRK1A gene dosage and activity. As a result of their co-translational activation loop phosphorylation the DYRKs are active once translated, however, there is growing evidence that some DYRKs are subject to additional post-translational modification and regulation. Autophosphorylation of DYRK1A allows binding of 14-3-3, which promotes DYRK1A catalytic activity [17,18]. In C. elegans, the DYRK homologue, minibrain kinase-2, is activated during oocyte maturation by cyclin-dependent kinase-1 (CDK1)-dependent phosphorylation of serine 68, a residue outside of the kinase domain that is required for full activity in vivo [19]. DYRK2 is phosphorylated at T33 and S369 (numbering corresponding to the short form of DYRK2) by the Ataxia telangiectasia mutated kinase (ATM) in response to genotoxic stress; this inhibits the ubiquitination of DYRK2, which can then phosphorylate S46 of the tumour suppressor p53 [20]. Finally, mass spectrometry of human DYRK4 expressed in HEK293 cells has identified phosphorylated Ser and Thr residues, indicating that DYRK4 is phosphorylated by other cellular protein kinases [21].
DYRK1B is implicated in promoting differentiation in several models: for example, it is expressed de novo during myogenesis [22] and undergoes differential splicing during adipogenesis [23]. Indeed, DYRK1B can promote cell cycle arrest by multiple mechanisms including promoting cyclin D1 (CCND1) degradation [24,25] by direct phosphorylation at T286 [25] and increasing the expression of the cyclin-dependent kinase inhibitors p21 CIP1 and p27 KIP1 [24,25]. Mutations in DYRK1B have been reported in an inherited form of metabolic syndrome associated with early-onset coronary artery disease, obesity, hypertension and diabetes [26]. In addition, DYRK1B is amplified [27,28] and mutated [29] in certain cancers and has been reported to promote cell survival [30][31][32]. Despite this, less is known about the post-translational regulation of DYRK1B. It has been suggested that oncogenic KRAS stimulates DYRK1B kinase activity [33] and that DYRK1B is a downstream effector of KRAS [34] but the molecular details of this regulation remain unclear.
There is growing interest in inhibiting DYRKs and several small molecule inhibitors of the class I DYRKs have been described including DYRK1B-selective inhibitors such as AZ191 [25] and dual 1A/1B inhibitors such as INDY [35] and Harmine [36]. These inhibitors are selective for the Ser/Thr kinase activity of the mature DYRKs [25,36], only inhibiting the Tyr kinase activity at very high doses, and are, therefore, useful in helping to define DYRK substrates and DYRK functions. To progress our interest in DYRK1B, we sought to identify DYRK1B autophosphorylation sites that were dependent on the Ser/Thr kinase activity of mature DYRK1B, since these might serve as biomarkers for DYRK1B activity and DYRK1B inhibitors. Here, we identify serine-421 (S421) as a site of DYRK1B trans-autophosphorylation that is inhibited by DYRK1B Ser/Thr kinase inhibitors and contributes to DYRK1B kinase activity. Remarkably, S421 is also phosphorylated in a MEK1/2-ERK1/2-dependent fashion in cells and by ERK2 in vitro, defining DYRK1B as a new substrate of ERK2 and providing a link between between two kinases involved in cell fate decisions. Finally, we show that mutations in DYRK1B that are found in cancer or metabolic syndrome either inhibit or are without effect on DYRK1B kinase activity. These results provide new insights into the regulation and role of DYRK1B.

Plasmids
For the mass spectrometry experiments, DYRK1B sequence (Swissprot Q9Y463; Embl AF205861) was ordered from Geneart with a c-terminal myc-tag and subcloned into pcDNA3. pcDNA3-DYRK1B was mutated by site-directed mutagenesis to generate catalytically inactive Y271F/Y273F and K140R constructs. For other experiments DYRK1B was amplified by PCR from pOTB7-DYRK1B (from Mammalian Gene Collection) and subcloned into pCMV-Tag2B and pEGFP-C3. pCMV-Tag2B-DYRK1B was mutated by site-directed mutagenesis to generate to catalytically inactive DYRK1B (D239A). pCGN-KRAS12V was from Adrienne Cox, Department of Pharmacology, University of North Carolina at Chapel Hill. The sequences of all oligos used are available upon request.

Analysis of in vitro autophosphorylation of recombinant DYRK1B by mass spectrometry
Recombinant DYRK1B expressed in insect cells (Carna Biosciences) was allowed to autophosphorylate in the presence of ATP. The protein was then resolved on a Novex gel followed by Coomassie staining. The Coomassie bands were reduced with DTT (Sigma), alkylated with iodoacetamide (Sigma), followed by digestion with: trypsin only; AspN and trypsin; chymotrypsin only; chymotrypsin and trypsin (all enzymes supplied by Roche). The resultant peptides were extracted and concentrated. The sample was chromatographed using the Dionex U3000 nanoflow chromatography system (ThermoFisher), the outlet flow ran directly into the Qstar Elite mass spectrometer (ABSciex) for analysis via the nanoflow probe at a flow rate of 300 nl/min. A 40 min reversed phase gradient was run using a 300 lm i.d. 9 5 mm C18 PepMap (Dionex ThermoFisher) trapping pre-column and a 75 lm i.d. 9 15 cm C18 ACE analytical column (Hichrom). The Qstar collected data in positive ion mode and an auto-switching setup was initiated with automatic precursor selection based on peak intensity and charge state. The subsequent data files generated were searched against the Swissprot database, using the Mascot Daemon software. The searches were then manually verified to confirm phosphorylation sites. From the MSMS data of the phosphorylated and nonphosphorylated peptides, the parent mass and key daughter ion masses were used to generate an MRM (multiple reaction monitoring) method on the 4000 Qtrap mass spectrometer (ABSciex).

Analysis of DYRK1B autophosphorylation in recombinant enzyme and cells by mass spectrometry using MRM
Recombinant DYRK1B enzyme was incubated with ATP in the presence or absence of 10 lM AZ'294 (an early DYRK1B HTS hit, with an IC 50 of \ 0.03 lM in an in vitro enzyme assay), and run on a Novex gel followed by Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity… 885 Coomassie Staining. Reduction, alkylation and digestion with trypsin was carried out as above. Wild-type or mutant myc-DYRK1B proteins were transiently expressed in COS-1 cells. DYRK1B proteins were enriched from whole cell lysates using anti-myc antibody, after treatment with 10 lM AZ'294 or 0.1 % DMSO for 2 h. Immuno-complexes were digested with trypsin prior to nano-liquid chromatography mass spectrometry. MRM was performed on the 4000 Qtrap mass spectrometer using the previously generated method from the recombinant analysis. The sample was chromatographed using the Dionex U3000 nanoflow chromatography system (ThermoFisher) the outlet flow ran directly into the 4000 Qtrap for analysis via the nanoflow probe at a flow rate of 700 nl/min. A 5 min reversed phase gradient was run using a 300 lm i.d. 9 5 mm C18 PepMap (Dionex ThermoFisher) trapping precolumn and a 75 lm i.d. 9 15 cm C18 ACE analytical column. The column was then equilibrated culminating in a 12.5 min run time per sample. The non-phosphorylated and phosphorylated masses of the pS421 containing tryptic peptide were monitored. Peak quantification was established using ABSciex software, this was manually verified and exported to Excel for further data analysis.

Preparation of cell extracts and Western blotting
Cells were lysed in ice-cold TG lysis buffer, assayed for protein content and fractionated by SDS-PAGE as described previously [38]. SDS-PAGE gels were transferred to Immobilon P membranes (Millipore), which were blocked in 0.1 % (v/v) Tween-20-TBS (tris-buffered saline) containing 5 % (w/v) powdered milk and probed with the indicated antibodies. Immune complexes were visualised with the ECL system (GE Healthcare).
In vitro DYRK kinase assay FLAG-DYRK1B [wild-type (WT), kinase-dead (KD, D239A) or S421A (SA)] were immunoprecipitated from whole cell lysates with anti-FLAG antibodies and then assayed for kinase activity by incubating with 50 lM Woodtide with two additional lysines attached to the N terminus to allow it to bind to P81 paper (KKISGRL-SPIMTEQ), 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM MgCl 2 , 0.1 mM [c-32 P]ATP in a total volume of 50 ll for 20 min at 30°C as described previously [7]. For each experiment, a single IP was used to generate three technical replicates of 32   The transfection mixes were incubated at room temperature for 20 min to allow the formation of siRNA complexes. Cells were seeded the previous day in Pen/ Strep-free medium to achieve a confluency of 50 % on the day of transfection. 500 ll of the transfection solution was added to each well of the tissue culture plate and the cells were incubated 37°C in a humidified incubator with 5 % (v/v) CO2 for 16 h. After this 16 h, the medium was changed to fresh Pen/Strep-free media containing DMSO or 1 lM Selumetinib. The cells were incubated for a further 48 h at 37°C in a humidified incubator with 5 % (v/v) CO2 prior to harvesting.

Analysis of cell cycle distribution and cell death
Cell cycle distribution (G 1 , S, G 2 /M) and the fraction of dead cells (those with sub-G1 DNA content) was assessed by staining fixed cells with propidium iodide followed by flow cytometry as described previously [38,39].

Results
Identification of S421 as a novel DYRK1B autophosphorylation site that promotes kinase activity When expressed in cells catalytically inactive DYRK1B exhibits enhanced migration on SDS-PAGE gels compared to wild-type suggesting that DYRK1B normally undergoes autophosphorylation in vivo [25]. Activation of DYRK1B requires auto-phosphorylation in cis at tyrosine 273 (Y273) within the activation loop. However, once translation is complete mature DYRK1B phosphorylates substrates on Ser/Thr residues in trans. We wished to develop an assay to monitor the kinase activity of mature DYRK1B in cells, hoping that this might serve as a biomarker to support the development of DYRK1B inhibitors. To determine if DYRK1B could autophosphorylate on other residues, we analysed potential auto-phosphorylation sites on recombinant DYRK1B by proteolytic digestion and mass spectrometry (MS). Using a combination of endoproteases we achieved 85 % coverage of DYRK1B and identified twelve phosphorylation sites including p-Y63 and p-Y273 that have been previously reported (Supplementary Table 1). A targeted MS method was developed to quantify these phosphorylation sites in Cos1 cells expressing Myc-DYRK1B using multiple reaction monitoring (MRM). This analysis identified four phosphorylation sites in cells but only one, S421 in the peptide IpSPLGALQHGFFR, was inhibited when cells were incubated in the presence of AZ'294, an early stage precursor of the DYRK1B inhibitor AZ191 [25] (Table 1). Cellular p-S421 was almost completely abolished when catalytically inactive DYRK1B mutants were expressed in Cos1 cells, including the activation loop mutant DYRK1B Y271F/Y273F or the catalytic lysine mutant DYRK1B K140R (kinase-dead or KD); these results demonstrated that p-S421 in cells was strongly dependent on DYRK1B kinase activity (Fig. 1a).
To progress this analysis further a p-S421 phosphospecific antibody was raised in rabbits and validated by its ability to detect wild-type DYRK1B but not the nonphosphorylatable DYRK1B S421A mutant when they were both expressed in HEK293 cells (Fig. 1b). Treatment of HEK293 cells with either of two DYRK1B inhibitors, AZ191 [25] or harmine [36], strongly inhibited p-S421 demonstrating that DYRK1B kinase activity was required for efficient S421 phosphorylation in cells (Fig. 1c). In contrast, we have previously shown that these inhibitors do not inhibit auto-phosphorylation at tyrosine, representing p-Y273 and p-Y63 [25], demonstrating that at the doses used they selectively inhibited the Ser/Thr kinase activity of mature DYRK1B. Since S421 is located in the DYRK1B kinase domain we determined if S421 phosphorylation had any role in DYRK1B kinase activity by comparing wild-type and DYRK1B S421A in kinase assays. DYRK1B proteins were expressed in HEK293 cells and isolated by immunoprecipitation prior to being assayed for kinase activity in vitro; kinase activity was normalised to the amount of wild-type or mutant DYRK1B that was recovered in the immunoprecipitation. These assays revealed that DYRK1B S421A exhibited a significant 50 % reduction in intrinsic kinase activity compared to wild-type DYRK1B (Fig. 1d) whereas kinase-dead DYRK1B D239A (KD) was completely defective. However, since only *50 % of DYRK1B was The resultant immuno-complexes were divided into two aliquots. The first aliquot was assayed for kinase activity against the synthetic substrate peptide Woodtide (50 lM) (upper panel). The second aliquot was separated by SDS-PAGE gels and transferred onto PVDF membrane. The amount of DYRK1B protein in each sample was determined by quantitative analysis of the immunoblot using Licor Odyssey (lower panel). DYRK1B kinase activity levels were normalised to the amount of DYRK1B present in each immunoprecipitate. Data were averaged from five separate experiments each with three technical replicates with each bar representing the mean ± standard deviation. Statistical analysis was performed using a two-tailed paired t test phosphorylated at S421 when over-expressed (Fig. 1a), this is probably an underestimate of the contribution of S421 to DYRK1B kinase activity. Thus, S421 phosphorylation contributes to DYRK1B kinase activity.

DYRK1B autophosphorylation at S421 occurs in trans
Based on the current model of DYRK activation [4,7], DYRK1B autophosphorylation of Y273 is thought to occur in cis through an intramolecular mechanism. To investigate if S421 auto-phosphorylation also occurred in cis, we expressed discrete DYRK1B proteins of different sizes (using either EGFP-DYRK1B or FLAG-DYRK1B) so that we could distinguish S421 phosphorylation in cis or in trans (Fig. 2a). FLAG-DYRK1B D239A (KD) expressed alone was not phosphorylated on S421, nor was wild-type EGFP-DYRK1B able to phosphorylate FLAG-DYRK1B S421A (SA). However, both EGFP-and FLAG-tagged wild-type DYRK1B auto-phosphorylated on S421, and this was blocked by AZ191 or harmine. Finally, FLAG-DYRK1B D239A (KD) was phosphorylated on S421 when co-expressed with wild-type EGFP-DYRK1B and this was blocked by AZ191 or harmine, suggesting this was an intermolecular phosphorylation (Fig. 2a). In addition, we expressed two different kinase-dead DYRK1B mutants-DYRK1B K140M or DYRK1B D239A -in HEK293 cells, immunopurified them and then incubated them in vitro with purified recombinant GST-DYRK1B; this is a far simpler system than co-expression in cells (Fig. 2a). These experiments demonstrated that purified GST-DYRK1B could indeed phosphorylate the two different kinase-dead DYRK1B mutants in vitro (Fig. 2b) and in both cases this in vitro trans-autophosphorylation was abolished by AZ191. Whilst we cannot rule out the potential involvement of an accessory protein co-purifying with the DYRK1B mutants, these results strongly suggest that one DYRK1B molecule can directly phosphorylate another on S421 in vitro. Recombinant GST-DYRK1B was also able to autophosphorylate at S421 under these conditions and this was again inhibited by AZ191. In summary, these data strongly suggest that S421 is a novel site of DYRK1B auto-phosphorylation that can proceed through an in trans mechanism.
Activation of the RAF-MEK-ERK1/2 signalling pathway also promotes S421 phosphorylation in cells In the course of this work we noted that whilst the DYRK1B S421A mutant was completely defective for p-S421 in cells (Fig. 1b), ATP-competitive inhibitors of DYRK1B Ser/Thr kinase activity left some residual p-S421 signal (Fig. 1c), raising the possibility that other cellular protein kinases might contribute to S421 phosphorylation. To investigate this we co-expressed DYRK1B together with an activated KRAS G12V mutant because: (i) KRAS drives activation of multiple protein kinase cascades and (ii) expression of activated KRAS has previously been proposed to activate DYRK1B by up to 20-fold [33]. Wildtype DYRK1B or kinase-dead DYRK1B D239A (KD) was transiently expressed with KRAS G12V for 24 h in HEK293 cells. DYRK1B proteins were then isolated from whole cell extracts by immunoprecipitation and assayed in an in vitro DYRK1B kinase assay; activity was again normalised to the amount of DYRK1B that was recovered in the immunoprecipitation (Fig. 3a). DYRK1B kinase activity was increased by co-expression with KRAS G12V ; however, this effect was modest and not statistically significant, reflecting that fact DYRK1B was already very active when expressed in HEK293 cells. Consistent with this, KRAS expression also failed to significantly increase S421 phosphorylation over the already high basal level (Fig. 3b). As a control, DYRK1B D239A (KD) exhibited negligible kinase Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity… 889 activity that was unaffected by KRAS G12V expression. Control blots confirmed that KRAS G12V was expressed and active since it activated endogenous ERK1/2 (Fig. 3b).
During these experiments, we observed that kinase-dead DYRK1B D239A (KD), which cannot auto-phosphorylate on S421 (Fig. 1c), became phosphorylated on S421 when coexpressed with KRAS G12V (Fig. 3b). This suggested that S421 could also be phosphorylated by a kinase other than DYRK1B in cells and that this kinase was activated by KRAS G12V . KRAS activates multiple effector pathways including the RAF-MEK1/2-ERK1/2 pathway and various PI3K-dependent pathways. However, we noted that S421 is immediately followed by a proline residue (P422) in a Ser-Pro motif, the minimum required for phosphorylation by proline-directed kinases such as ERK1/2. To address if activation of the ERK1/2 pathway alone could increase p-S421, DYRK1B D239A (KD) was expressed in HR1 cells [39]; these cells are derived from HEK293 cells and harbour the DCRAF:ER* fusion protein (Fig. 4a) that is activated by 4-hydroxytamoxifen (4HT). Upon activation of DCRA-F:ER*, DYRK1B D239A (KD) became phosphorylated on S421 and this was blocked by the highly selective MEK1/2 inhibitor, selumetinib (AZD6244/ARRY-142886) [40] (Fig. 4b). To investigate if S421 phosphorylation following RAF activation also occurred on wild-type DYRK1B, we used HD1B cells, a cell line that harbours DCRAF:ER* and exhibits tetracycline-inducible expression of DYRK1B [25]. When DYRK1B expression was induced with tetracycline, the DYRK1B protein displayed autophosphorylation on S421 (Fig. 4c). Treatment of these cells with the DYRK1B inhibitor AZ191 blocked p-S421 autophosphorylation but S421 phosphorylation was restored by DCRAF:ER* activation and this was again blocked by selumetinib. Thus, activation of the ERK1/2 signalling pathway was sufficient to drive p-S421 of DYRK1B in cells.

ERK2 directly phosphorylates DYRK1B on S421 in cells and in vitro
If DYRK1B is a direct substrate of ERK1/2 then the kinetics of S421 phosphorylation on DYRK1B should closely follow the kinetics of ERK1/2 activation in cells and should also match the phosphorylation of bona fide ERK1/2 substrates such as BIM EL [42] and RSK [43]. To address this we again expressed DYRK1B D239A (KD) in HR1 cells and stimulated them with 4HT for various times Wild-type or kinase-dead DYRK1B were transiently expressed together with empty vector or KRAS G12V for 24 h in HEK293 cells. a FLAG-DYRK1B proteins were isolated from whole cell extracts by immunoprecipitation and immuno-complexes were divided into two aliquots. The first aliquot was separated by SDS-PAGE, transferred onto PVDF membrane and immunoblotted for DYRK1B. DYRK1B levels were quantified on Licor Odyssey (upper panel). Data are from a single experiment representative of five separate experiments. The second aliquot was incubated with the synthetic DYRK substrate peptide Woodtide in an in vitro kinase assay to determine kinase activity. In vitro kinase activity was normalised to the amount of DYRK1B proteins present on the immuno-blot (lower panel). Data in the graph are presented as mean ± standard deviation from five separate experiments each with three technical replicates. Statistical analysis was performed using a two-tailed paired t test. b Whole cell extracts were fractionated by SDS-PAGE, transferred onto PVDF membrane and immuno-blotted with the indicated antibodies. Data are from a single experiment representative of five separate experiments with similar results to activate DCRAF:ER*. Activation of ERK1/2 was observed as early as 15 min after 4HT addition and persisted for the full 120 min timecourse (Fig. 5a). Increases in p-S421 DYRK1B tracked precisely with p-ERK1/2, being apparent from 15 min onwards; furthermore, inclusion of selumetinib at each timepoint abolished ERK1/2 activation and the p-S421-DYRK1B signal (Fig. 5a). In addition, phosphorylation of S421 on DYRK1B precisely matched the MEK1/2-dependent phosphorylation of RSK and BIM EL (monitored by it's characteristic reduction in mobility on SDS-PAGE) following activation of DCRAF:ER*. To determine if ERK2 could directly phosphorylate DYRK1B at S421, recombinant, active GST-ERK2 was incubated with FLAG-DYRK1B in an in vitro kinase assay (Fig. 5b). In this assay, wild-type DYRK1B was able to auto-phosphorylate on S421 in the absence of AZ191 (Lane 2) as shown by 32 P incorporation and pS421-DYRK1B western blot. However, 32 P was still incorporated into DYRK1B S421A in the absence of AZ191 (Lane 6), albeit to a lesser extent than wild-type DYRK1B, suggesting that there are other sites of DYRK1B auto-phosphorylation in vitro in addition to S421, consistent with our MS analysis (Table 1, Supplementary Table 1). In the presence of AZ191, DYRK1B auto-phosphorylation was blocked (Lanes 3 and 7). When active ERK2 was added to DYRK1B in the presence of AZ191, DYRK1B became phosphorylated on S421 (Lane 4) as shown by 32 P incorporation and p-S421 western blot and this was blocked by the ERK1/2 inhibitor SCH772984 [44] (Lane 5) demonstrating that S421-DYRK1B was a direct substrate of ERK2 in vitro. Interestingly, ERK2 was also able to phosphorylate DYRK1B S421A (Lane 8) although to a lesser degree, indicating that ERK2 may also be able to phosphorylate other sites on DYRK1B in vitro. Taken together these results demonstrated that in addition to auto-phosphorylation, DYRK1B is also phosphorylated on S421 by ERK1/2 in cells and in vitro.

Inhibition of MEK1/2-ERK1/2 signalling by selumetinib promotes DYRK1B expression
It has previously been show that first generation, pan-MEK1/2/5 inhibitors PD98059 and U0126 can increase DYRK1B mRNA and protein [22,45]. Notwithstanding the off-target effects of these drugs these results suggest that ERK1/2 signalling may normally repress DYRK1B expression. Our demonstration that ERK1/2 phosphorylates DYRK1B at S421 prompted us to examine DYRK1B expression using the more selective, second generation MEK1/2 inhibitor selumetinib [40]. Indeed, when we treated the melanoma cell line MelJuso (HRAS G13D / NRAS Q61L ) with 1 lM selumetinib this strongly and immediately inhibited ERK1/2 signalling and increased the expression of DYRK1B after a delay of 8 h. This was accompanied by an increase in DYRK1B mRNA which was apparent after 5 h of selumetinib treatment (Supplemental Fig. 1a). Treatment with actinomycin D, an inhibitor of transcription, prevented the increase in DYRK1B expression observed in response to selumetinib treatment (Supplemental Fig. 1b) suggesting that the increase in DYRK1B expression observed upon MEK inhibition reflects de novo gene transcription. Finally, similar results were obtained in the A375 melanoma cell line (BRAF V600E ) (Supplemental Fig. 1c), where selumetinib caused a dose-dependent inhibition of ERK1/2 signalling and an increase in expression of DYRK1B as well as BIM EL , which is known to be repressed by the ERK1/2 pathway [46]. Since inhibition of ERK1/2 Empty vector (EV), FLAG-DYRK1B wild-type or FLAG-DYRK1B S421A were transiently expressed in HEK293 cells. 4 h post-transfection, cells were treated with 10 lM AZ191 for a further 24 h to minimise DYRK1B autophosphorylation on S421. FLAG-DYRK1B proteins were then isolated from whole cell extracts by anti-FLAG immunoprecipitation and the resulting immuno-complexes were washed and used as a substrate in an ERK2 in vitro kinase assay in the presence of [c-32 P]ATP as described in ''Materials and methods''. Some of the assay tubes included the ERK1/2 inhibitor SCH772984. The terminated assay reactions were separated by SDS-PAGE, transferred onto PVDF membrane and 32 P incorporation was detected by autorad. Subsequently, the membrane was blotted with the indicated antibodies. Data are from a single experiment representative of two separate experiments with similar results signalling by selumetinib typically causes a G1 cell cycle arrest [47] (Fig. 6b), the increase in DYRK1B expression could simply be a consequence of cell cycle arrest rather than a specific consequence of reduced ERK1/2 signalling. To address this, we compared the effect of selumetinib with that of palbociclib (PD-0332991), a potent and highly selective inhibitor of CDK4 and CDK6 [48]. Whilst both drugs elicited a strong G1 cell cycle arrest (Fig. 6b) and inhibited phosphorylation of RB (Fig. 6c), only selumetinib inhibited ERK1/2 signalling and increased DYRK1B expression (Fig. 6c), demonstrating that the increase in DYRK1B was not simply a consequence of cell cycle arrest. Thus ERK1/2 signalling provides opposing regulatory inputs into DYRK1B: ERK1/2 can directly phosphorylate DYRK1B at S421, a residue that promotes kinase activity, but ERK1/2 signalling also represses DYRK1B expression.
DYRK1B has been proposed to function as a pro-survival protein kinase [30][31][32]. Since ERK1/2 inhibition by selumetinib typically results in a G1 cell cycle arrest with little cell death [47] we considered the possibility that DYRK1B expression might provide a survival signal following MEK1/2-ERK1/2 inhibition, thereby preventing cell death; that is, combined inhibition of MEK1/2 and DYRK1B would be synthetic lethal. To test this we treated MelJuso cells with selumetinib alone or in combination with the DYRK1B inhibitor AZ191 or in combination with DYRK1B siRNA, with GFP siRNA as a control (Fig. 7a). AZ191 treatment alone had no effect on cell death as judged by PARP cleavage (Fig. 7a) and the fraction cells with hypodiploid (sub-G1) DNA (Fig. 7b). Selumetinib treatment caused only a small increase in PARP cleavage and cell death. The combination of AZ191 and selumetinib caused greater cell death than either drug alone; however, whilst this was statistically significiant, it was only a very modest effect. We also observed a modest increase in cell death when we combined selumetinib with DYRK1B siRNA; however, this was not statistically significant (Fig. 7c). Finally, despite the induction of DYRK1B, we failed to observe any increase in cell death when selumetinib was combined with AZ191 or DYRK1B siRNA in A375 cells (data not shown). Thus, any synthetic lethal relationship between DYRK1B and MEK1/2 was at best weak and variable between cell lines.
Mutants of DYRK1B found in cancer or metabolic syndrome exhibit normal or reduced kinase activity Systematic sequencing of cancer genomes has revealed rare mutations in DYRK1B including L28P, R102H, S234G and Q275R [29]. However, the low incidence of these mutations calls into question whether they are 'drivers' or 'passengers' and no further functional information has been forthcoming. In addition, mutations in DYRK1B have been found in familial cases of metabolic syndrome [26,49] and include: gain-of-function mutations R102C and H90P; putative loss-of-function mutations, such as L28P, and apparently benign mutations, such as G352A and P578S. However, the impact of these disease-associated mutations on intrinsic kinase activity has not been described. To address this directly we engineered some of these mutations into individual DYRK1B constructs, expressed them in HEK293 cells, isolated them by immunoprecipitation and assayed their kinase activity in vitro. Our results revealed a consistent pattern: DYRK1B L28P (found in rare cases of metabolic syndrome as well as cancer) exhibited identical kinase activity to wild-type DYRK1B; DYRK1B R102H and DYRK1B S234G exhibited 40-50 % reduction in kinase activity and DYRK1B Q275R exhibited a 90 % reduction in kinase activity (Fig. 8a). We also directly compared the activity of the R102H mutant (found in cancer) and the R102C mutant (found in metabolic syndrome); both mutants exhibited a 50-60 % reduction in kinase activity (Fig. 8a). Finally, the relative activity of these DYRK1B mutants correlated well with their degree of phosphorylation at S421 (Fig. 8b) suggesting that in the absence of strong inputs from the ERK1/2 pathway p-S421 correlated well with intrinsic activity. Thus mutations in DYRK1B that are found in cancer (L28P, R102H, S234G and Q275R) or metabolic syndrome (L28P, R102C) have either no effect on intrinsic kinase activity or reduce kinase activity, in one case (DYRK1B Q275R ) severely.

Discussion
Trans-autophosphorylation at S421 contributes to DYRK1B activity Our results identified S421 as a site of DYRK1B autophosphorylation since it was present in purified recombinant wild-type DYRK1B incubated in vitro with ATP, absent in kinase-dead DYRK1B and strongly (though not completely) inhibited by DYRK1B inhibitors in vitro or in cells. These drugs do not prevent autophosphorylation on tyrosine (largely Y273, but also Y63) in cells [25] except at high concentrations [36], consistent with phosphorylation of Y273 and S421 proceeding through different mechanisms. Indeed, we have now shown that S421 autophosphorylation can proceed through an intermolecular trans-phosphorylation mechanism whereas Y273 is an intramolecular cis-phosphorylation event. To our knowledge, this is the first description of a serine or threonine auto-phosphorylation site on DYRK1B. S520 in DYRK1A was previously identified as a site of auto-phosphorylation that was required for full DYRK1A kinase activity [18]; in this case S520 auto-phosphorylation increased 14-3-3b binding to DYRK1A. However, phosphorylation of S520 in DYRK1A took place in cis, in contrast to the intermolecular phosphorylation of S421 in DYRK1B described here. Indeed, given the position of S421, situated away from the kinase active site, it is difficult to imagine it could be phosphorylated in cis through an intramolecular mechanism. Regardless, it seems that S421 phosphorylation also contributes to DYRK1B kinase activity, either directly or by an indirect mechanism involving accessory proteins, since the S421A mutant exhibited a consistent 50 % reduction in kinase activity; indeed, this is likely to be an underestimate of the impact of the S421A mutant since only half of over-expressed DYRK1B was phosphorylated at S421. Whilst a twofold modulation of kinase activity may seem modest, it is important to remember that even a 50 % increase in expression and activity of the closely related DYRK1A has profound physiological effects in mice and phenocopies some Down's syndrome defects [11,  15] whilst a 50 % decrease in DYRK1A causes even more dramatic effects in Drosophila, mice and human patients including microcephaly, growth retardation and behavioural defects [11,50,51]. S421 occurs close to the highly conserved arginine residue (AARIS 421 PL) shortly after alpha helix H/kinase sub-domain XI [3,10]. Serine and threonine are the first and third most common residues at this position across the kinome as a whole, whilst the Cancer Genome Atlas (TCGA) reveals that the position equivalent to S421 is ranked 25 th in terms of frequency out of the 2500 identified mutation positions across kinase domains, suggesting a potential wider significance for phosphorylation at this site. Of the 61 human CMGC kinases listed at http://kinase.com/human/kinome/groups/cmgc.aln only 7 lack a Ser or Thr at the position corresponding to Ser421 of DYRK1B. More significantly, S421 in DYRK1B is a Pro-directed site (Ser-Pro) and within the closely related human DYRK family (DYRK1A, 1B, 2, 3 & 4 and the four HIPKs) the corresponding site is a Ser-Pro or Thr-Pro in all cases except DYRK1A (Gln-Pro) raising the possibility that these kinases may also be regulated by phosphorylation at this site. This site is also Ser-Pro or Thr-Pro in DYRKs from lower eukaryotes including S. pombe Pom1p, S. cerevisiae Yak1p, C. elegans MBK-1 and MBK-2 and D. melanogaster dDYRK2 and dDYRK3, indicating evolutionary conservation of the site. In terms of functional relevance, intermolecular phosphorylation at S421 suggests that DYRK1B may be able to form dimers. Indeed, the structure of the closely related DYRK1A [10] revealed multiple copies per unit cell with Q469 of DYRK1A, corresponding to S421 of DYRK1B, well away from the ATP binding pocket at the interface between DYRK1A dimers. It is currently unclear if DYRK1B is a dimer in cells or whether it is more or less active as a dimer or monomer but it will be interesting to determine if S421 phosphorylation influences dimerisation. Alternatively, S421 phosphorylation may influence substrate interactions, which would be consistent with the reduction in kinase activity that we observe for the S421A mutant. For example, the closely related DYRK4 exhibits an alternative splicing donor site in exon 19 which removes three amino acids (DCLV) at the end of the a-helix H, immediately prior to the conserved Arg in subdomain XI [21]. This region is thought to contribute to the substrate-binding structure [52] and it is notable that DYRK4 DCLV exhibits reduced kinase activity against exogenous substrates compared to the full length DYRK4 [21]. Thus, it is possible that changes to this region in DYRK1B, such as phosphorylation of S421, might contribute to ordering of the substrate-binding structure. Such speculation will need to be addressed by future structural studies.

ERK1/2 can phosphorylate DYRK1B at S421
We sought to identify DYRK1B autophosphorylation sites as biomarkers for DYRK1B kinase activity and identified S421 as a phosphorylation site that promotes DYRK1B activity. However, we found that S421 is also phosphorylated by ERK1/2; whilst this disqualifies S421 as a unique DYRK1B activity biomarker it reveals a new mode of cross talk between ERK1/2 and DYRK1B.
We noted that inhibitors of DYRK1B Ser/Thr kinase activity left a residual p-S421 signal in cells, prompting a search for other kinases that might target this site. For this purpose we co-expressed DYRK1B with KRAS G12V , since KRAS has several effectors (PI3K, RAL, etc.,) and activates multiple protein kinase signalling pathways. In contrast to previous reports of a 9-to 20-fold activation [33] we consistently found that KRAS G12V caused only a very modest (10-15 %) increase in activity that was not statistically significant. This was not due to a deficit in our KRAS construct as KRAS G12V strongly activated endogenous ERK1/2 in the same transfections. These results suggest that any effect of activated KRAS on DYRK1B effector pathways such as Hedgehog [34] may be independent of DYRK1B intrinsic kinase activity, though we cannot rule out a possible effect on DYRK1B substrate binding (see above) that might be missed in an assay that employs simplified peptide substrates. However, these experiments also revealed that KRAS G12V could actually increase DYRK1B phosphorylation at S421. Through a combination of conditional kinases and selective kinase inhibitors we were able to show that ERK1/2 activation alone could increase p-S421, commensurate with established ERK1/2 substrates, and ERK2 could phosphorylate S421 in vitro. This is the first report to clearly define DYRK1B as a substrate of ERK2. A previous study suggested that ERK2, JNK or p38 might phosphorylate DYRK1B but stopped short of specifying the kinase, identifying the phosphorylation site or the effect on DYRK1B function [53].
Together with previous studies, our results reveal a complex interplay between ERK1/2 signalling and DYRK1B. Since phosphorylation of S421 may enhance DYRK1B kinase activity, ERK1/2 inhibition could decrease DYRK1B activity. In contrast, previous studies have indicated that inhibition of ERK1/2 signalling may increase DYRK1B expression [22,45] and we also observed this with the highly selective MEK1/2 inhibitor selumetinib, suggesting that ERK1/2 signalling can repress DYRK1B expression. Thus, ERK1/2 inhibition may reduce the activity of existing DYRK1B but also increase de novo DYRK1B expression, providing a mechanism for fine tuning DYRK1B activity. It is well known that the ERK1/2 pathway is a key regulator of the cell cycle, promoting proliferation or cell cycle arrest depending on the magnitude or duration of ERK1/2 activity [54]. DYRK1A and DYRK1B have also emerged as regulators of the cell cycle; both can promote the turnover of CCND1, increase expression of p21 CIP1 and/or p27 KIP1 and thereby promote cell cycle arrest and differentiation [22,24,25,45,55]. Indeed, even modest (*50 %) changes in DYRK1A expression and activity exert profound effects on the cell cycle [56]. ERK1/2-dependent regulation of DYRK1B adds another level of control, providing a link between two cell fate signalling pathways.
There is growing interest in identifying synthetic lethal drug combinations in the treatment of cancer [57]. For example, whilst selumetinib exerts a largely cytostatic effect, combining the BH3-mimetic ABT-263 with selumetinib causes up to 80 % cell death in colorectal cells and delays the onset of acquired resistance to selumetinib [47]. The increase in DYRK1B expression following MEK1/2-ERK1/2 inhibition prompted us to investigate whether inhibition of DYRK1B might similarly transform the cytostatic effects of selumetinib to promote cell death. Indeed, Gao et al. recently described a twofold increase in H292 cell death when the pan-MEK1/2/5 inhibitor U1026 was combined with DYRK1B siRNA [45], although they did not report the actual percentage of dead cells. We also observed a twofold increase in cell death in MelJuso cells when selumetinib was combined with DYRK1B siRNA or the AZ191 DYRK1B inhibitor. However, the percentage of dead cells observed under these conditions was modest, being less than 20 %, and was not observed in a second cell line (A375) that exhibited strong DYRK1B upregulation following ERK1/2 inhibition. Thus, DYRK1B and MEK1/ 2 do not seem to exhibit a robust synthetic lethal relationship that might form the basis for a rational drug combination.
DYRK1B mutants found in metabolic syndrome or cancer exhibit normal or reduced kinase activity DYRK1B is over-expressed due to gene amplification in certain tumours, including pancreatic cancer [27,28], and this has led to the suggestion that DYRK1B is a potential oncogene. In addition, cancer genome sequencing has revealed mutations in DYRK1B including L28P, R102H, S234G and Q275R [29]. Both L28 and R102 lie outside the DYRK1B kinase domain; L28 lies outside any known conserved regions whilst R102 is situated between the DYRK homology (DH) box and the kinase domain. S234 is conserved in the class 1 DYRKs and immediately precedes the catalytic loop in kinase subdomain VIb whilst Q275 is conserved in all DYRKs and follows the critical Y273 in the kinase activation loop within subdomain VIII. In addition, L28P and R102C mutations were found in cases of metabolic syndrome [26,49]. However, these diseaseassociated mutations were found at low frequency and their impact on DYRK1B activity was not assessed.
We found that these mutations were either neutral with respect to DYRK1B activity (L28P) or caused a consistent reduction in kinase activity (R102H, R102C S234G and Q275R); in the case of the Q275R mutation this effect was severe, with an approximate 90 % reduction in kinase activity. Q275 lies within the activation loop of DYRK1B; whilst activating, oncogenic mutations are found in this region at significant frequency in some protein kinases, mutations in this loop, including the introduction of bulky and highly positive side chain such as arginine, are also known to inhibit activity by impacting on activation loop conformation and flexibility. At this stage we cannot rule out the possibility that these mutations might confer kinase-independent properties upon DYRK1B; for example, by driving DYRK1B to a specific cellular location or engaging with particular partner proteins. Indeed, some of the effects of DYRK1B in metabolic syndrome were apparently independent of DYRK1B kinase activity [26]. Regardless, our results suggest that these DYRK1B mutations are unlikely to be gain-of-function in terms of intrinsic kinase activity and some may even be loss-offunction mutations. Since DYRK1B can promote cell cycle arrest and differentiation [22,24,25,45,55,56], such lossof-function mutations suggest that any oncogenic properties of DYRK1B are, at best, context-dependent; indeed, they may even suggest tumour suppressor functions for wild-type DYRK1B.
In summary, we demonstrate that phosphorylation of S421 contributes to DYRK1B kinase activity and that S421 is a site for DYRK1B autophosphorylation and phosphorylation by ERK1/2, defining DYRK1B as a new substrate of ERK1/2. Our results reveal opposing effects of ERK1/2 signalling on DYRK1B which may serve to fine tune DYRK1B activity. However, despite the ability of ERK1/2 to phosphorylate S421, KRAS G12V does not increase DYRK1B kinase activity. This may be because DYRK1B expressed in cells is already active so that any effects of KRAS are modest and incremental; this may require single cell analysis to resolve. Alternatively, different KRAS effectors may exert opposing effects on DYRK1B; so whilst activation of RAF-MEK-ERK may promote S421 phosphorylation, that would enhance activity, other KRAS effector pathways may exert opposing effects on DYRK1B. Indeed, our mass spectrometry analysis has identified at least two other phospho-Ser sites on DYRK1B expressed in cells that are not autophosphorylation sites; the kinases for these sites and the functional consequences remain unknown. Finally, mutants of DYRK1B found in cancer or metabolic syndrome are either neutral with respect to kinase activity or exhibit significant decreases in kinase activity. At least one of these, R102C in metabolic syndrome, is defined as a gain-of-function mutation [26] but clearly exhibits reduced kinase activity, raising the possibility that some effects of DYRK1B may not be strictly dependent on kinase activity.