No studies to date have elucidated the clinical factors associated with pedicle screw pull-out during or immediately after surgery. The aim of this study was to assess the frequency of pedicle screw pull-out by comparing intraoperative scans obtained using cone-beam computed tomography (CBCT) with postoperative scans obtained using computed tomography (CT). We also sought to determine the incidence of pedicle screw pull-out and identify relevant risk factors.This was a retrospective analysis of prospectively collected data for 742 pedicle screws placed in 76 consecutive patients who underwent at least triple-level posterior fixation for thoracic or lumbar spinal injury, spinal metastasis, or pyogenic spondylitis between April 2014 and July 2020. Pedicle screw pull-out distance in the axial and sagittal planes was compared between CT scans obtained 2 days postoperatively and CBCT images acquired intraoperatively. Risk factors associated with pedicle screw pull-out were investigated by multivariate logistic regression analysis.Pedicle screw pull-out was seen with 58 pedicle screws (7.8%) in 26 patients (34.2%). There were significant differences in age, number of fused segments, frequency of diffuse idiopathic skeletal hyperostosis (DISH), and medical history of osteoporosis for pedicle screw pull-out. Risk factors for pedicle screw pull-out were older age  and a diagnosis of DISH . Several cases suggest that use of connecting rods was an important factor in intraoperative pedicle screw pull-out.Our findings suggest that age, number of fused segments, presence of DISH, and medical history of osteoporosis are risk factors for pedicle screw pull-out, with the greatest being older age and DISH. Many authors have described the efficacy of posterior pedicle screw instrumentation , 2, whicNowadays, many institutions can perform spinal instrumentation surgery using intraoperative cone-beam computed tomography (CBCT) in a hybrid operating room , 7 or anLong instrumented fusion requires insertion of a large number of pedicle screws, and the above-mentioned intraoperative modalities help to ensure surgical safety. The position of the pedicle screws can be confirmed using intraoperative CBCT in a hybrid operating room after pedicle screw insertion before rod connection. We have encountered several cases of intraoperative pedicle screw pull-out as a result of rod connection during posterior thoracic or lumbar spine surgery. In long instrumented fusion in particular, pedicle screw pull-out was found to be a risk factor for pedicle screw loosening . It has In this study, we investigated the frequency of pedicle screw pull-out by comparing intraoperative CBCT images and postoperative CT images acquired 2 days after surgery. We also sought to identify and evaluate the relevant risk factors for screw pull-out.This study was approved by the Institutional Ethics Committee . In this study, we conducted a retrospective analysis of prospectively collected data from 76 consecutive patients who underwent long posterior spinal instrumented fixation at 3 or more levels for thoracic or lumbar spinal injury, spinal metastasis, or pyogenic spondylitis between April 2014 and July 2020. The patients were evaluated using CBCT intraoperatively in a hybrid operating room. Evaluation by CT was performed again on postoperative day 2 before mobilization. Demographic and surgical data collected included age, sex, body mass index (BMI), operating time, estimated blood loss, underlying disease, number of fused segments, use of a Hook system, PPS insertion, presence of diffuse idiopathic skeletal hyperostosis (DISH), medical history of osteoporosis, smoking, preoperative comorbidities, degree of sagittal alignment, alignment change in the sagittal plane, screw density, type of rod, and screw design.All patients underwent posterior spinal fixation in the prone position with the trunk on a radiolucent operating table in a hybrid operating room. Following anesthesia, standard surgical exposure was performed via a midline skin incision using an open approach. PPS placement was performed via a 1.5-cm stab incision made laterally. The targeting needle was inserted into the pedicle at the superolateral border based on the anterior-posterior view under rotational C-arm fluoroscopic guidance. The guidewire was inserted through the targeting device and into the pedicle. The pedicle was then tapped using the guidewire. Next, the pedicle screw was inserted over the guidewire. CBCT images were obtained at this timing to confirm the screw position. Finally, rods were installed aligned to each screw placed in the open surgery and were inserted starting from the most cranial skin incision in the PPS surgery. A new automatic rod bending system called the Bendini system  was not use in this study. The order in which the set-screws were installed varied among the operators , a 3D imaging tool that reconstructs projection data obtained by a rotational C-arm with a flat panel detector. The series consisted of 0.616-mm CBCT sections reconstructed at 0.616-mm intervals. Raw data were used to reconstruct axial 2.0-mm-thick CBCT sections every 2.0 mm with a field of view adequate for visualization of the spine, as well as sagittal and coronal reformatted images of the thoracolumbar spine. The position of the implant was assessed postoperatively on CT scans obtained using a 320-rowarea-detector CT system . The series consisted of 0.5-mm-thick CT sections reconstructed at 0.5-mm intervals. Raw data were used to reconstruct 2.0-mm-thick axial CT sections every 2.0 mm with a field of view that was adequate for spine visualization and for sagittal and coronal reconstruction of the thoracolumbar spine.Screw misplacement was classified according to the system devised by Schizas et al. . Screw mOther factors assessed were the degree of sagittal alignment between the upper instrumented vertebra (UIV) and lower instrumented vertebra (LIV) on intraoperative CBCT or postoperative CT, alignment change in the sagittal plane on intraoperative CBCT to postoperative CT Fig. , and scrU test, with statistical significance set at P < 0.05. Risk factors were determined using logistic regression analysis with a forward stepwise procedure (P < 0.1 for entry). The threshold of alpha and forward selection was chosen to stabilize the statistical model during stepwise covariate selection considering the relatively small number of our outcomes. Inter-observer agreement was measured using kappa coefficient scores. Any discrepancy between the observers was resolved by discussion. All statistical analysis was performed using JMP version 13.1.0 software .Statistical analysis was performed using Fisher’s exact test and the Mann-Whitneyn = 28, 36.8%), medical history of osteoporosis , rheumatoid arthritis , diabetes , and asthma . Three patients (3.9%) were on dialysis. A titanium rod was used in 60 cases (78.9%) and a cobalt chrome rod was used in 16 cases (21.1%). Mixed-thread screws were used in 15 cases (19.7%) and dual-thread screw were used in 61 cases (80.3%). Mean kyphotic angle of sagittal alignment between UIV and LIV was 6.2 ± 23.5 deg on intraoperative CBCT and 5.8 ± 23.5 deg on postoperative CT. Mean alignment change in the sagittal plane from intraoperative CBCT to postoperative CT was 2.1 ± 1.5 deg. Mean screw density was 1.69 ± 0.23 (screws/level).A total of 742 pedicle screws were inserted in the thoracic or lumbar spine of the 76 patients. Patient demographics are shown in Table Screw pull-out occurred for 58 of the 742 pedicle screws (7.8%) inserted, in 26 of the 76 patients (34.2%). For almost all cases of pull-out, insertion had been via PPS. The pedicle screw-related variables are shown in Table There was no significant difference in sex, BMI, operating time, estimated blood loss, underlying disease, use of a Hook system, surgical procedure, smoking status, preoperative comorbidities, degree of sagittal alignment, alignment change in the sagittal plane, screw density, type of rod, or screw design between the non-pull-out and pull-out groups. Patients in the pull-out group were more likely to be elderly, have more fused segments, have a diagnosis of DISH, and have a medical history of osteoporosis Table .The risk factors for pedicle screw pull-out were evaluated using logistic regression analysis. From univariate analysis, the dependent variable was defined as the presence of screw pull-out and the independent variables were age, number of fused segments, DISH, medical history of osteoporosis, and PPS procedure. As a result, the independent risk factors identified were older age  and DISH (odds ratio 3.35, 95% confidence interval 1.12–10.00, P = 0.0302; Table Previous reports have shown that pedicle screw loosening after surgery is a serious complication of spinal fixation surgery. Screw loosening causes nonunion back pain and sometimes neurological impairment, and it can be an indication for reoperation , 14. OhbIn our study, pull-out occurred for a total of 58 pedicle screws in 26 cases, giving an overall pedicle screw pull-out rate of 7.8%, which is relatively low compared with the rate of 16.2% reported previously . AlthougIn this study, patients in the pull-out group were more likely to be elderly, to have more fused segments, and to have a diagnosis of DISH or a medical history of osteoporosis. Other researchers have also identified older age and osteoporosis as risk factors for pedicle screw loosening , 15. TheLogistic regression analysis identified advanced presence of DISH to be independent risk factors for pedicle screw pull-out. DISH appears on radiographs and CT images as ossification along the anterolateral aspect of the vertebral bodies , 17. TheThe most important factor is to ensure appropriate rod bending and to gently connect the rod to each screw head. As it is sometimes difficult to create a perfect curve by manual bending, a new automatic rod bending system may be useful to improve the fitting of the rod . Also, aThere are several limitations in this study. First, we did not evaluate the screw length, diameter, position, or trajectory as factors affecting pull-out. Second, radiation exposure is a risk for patients, although we used intraoperative CBCT, in which the radiation exposure is reduced. Third, other important factors in preventing screw pull-out in the setting of in situ fusion are how perfectly the rod is contoured to the screws and how perfectly the head of each screw is aligned in the same plane. These technical issues could not be assessed with the present study design. Fourth, the sample size was limited; Nevertheless, this study identified age and presence of DISH as important risk factors for screw pull-out during surgery.This study analyzed the clinical factors associated with pedicle screw pull-out by comparing findings on both intraoperative CBCT and postoperative CT. Our findings suggest that age, number of fused segments, presence of DISH, and medical history of osteoporosis are risk factors for pedicle screw pull-out, with the greatest being older age and DISH.


CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation  distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4E343K, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4E343K) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4E343K causing WHIM in the reported family.Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor WT or CXCR4E343D (glutamic acid/E replaced with aspartic acid/D) or CXCR4E343K (glutamic acid/E replaced with lysine/K) or CXCR4E343R (glutamic acid/E replaced with arginine/R) or CXCR4E343A  and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4E343D had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4E343K or CXCR4E343R or CXCR4E343A had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor .We completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4Taken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.The online version contains supplementary material available at 10.1186/s12860-021-00347-9. Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) is a rare autosomal dominant inherited primary immunodeficiency disease (PID) –3. PatieCXCR4), which contains eighteen potential serine/threonine phosphorylation sites [E343K) at the 343 site of the CXCR4 tail, but no loss of serine and threonine residues for phosphorylation  is essential for the normal function of CXCR4, thus mentioning physiological events related with CXCR4. The charge-change substitution mutations  delay the desensitization and prolong down-stream signal transduction of CXCR4, and increase cell migration activities and aggravate inflammatory responses, thus causing pathogenic events, consequently leading to WHIM syndrome.E343K found in a family with WHIM syndrome does not introduce a loss of any single phosphorylation site in the C-terminus of CXCR4, but the mutation only is close to phosphorylation residues at S346/347 and S338/339. Biochemical analysis shows that the significant change of residue in CXCR4E343K is charge change, a substitution of the negative charge residues with positive residue (Glu/E to Lys/K), by which we made a speculation that the charge change alters the phosphorylation activities of the residues at S346/347 and S338/339 nearby, thus we created a series of mutations which cause different substitutions of the residue at 343 site of CXCR4 C-terminus, and established cell lines expressing those CXCR4 mutants, which has a substitution with either with a positive charge residue (e.g. Lys/K or Arg/R) or neutral residue (e.g. Ala/A for Glu/E), a negative charge residue (Asp/D for Glu/E), and systematically analyzed activities of those CXCR4 mutants in respect of their migration, phosphorylation, signaling pathway and immune response to clarify the mechanism underlying WHIM like mutation CXCR4E343K found in a family with WHIM syndrome at molecular level in vitro. Consistent with previous studies, our results show that CXCR4E343K mutation delays the receptor internalization and desensitization [E343K is a WHIM-like mutation. To further investigate downstream events of activated CXCR4 mutants, we detected the phosphorylation of ERK, p38, JNK, PI3K and AKT, and found that their phosphorylation in cells overexpressing CXCR4E343K was lasted longer than that in cells overexpressing CXCR4WT  or neutral charge (CXCR4E343A) replacement at the 343 site of receptor tail, cellular phenotypes of internalization and migration and immune responses are different from CXCR4WT and CXCR4E343D, the mutant with no charge change, given us that charge change at the 343site is the unique factor for the introduction of the activity abnormality of the mutants, CXCR4E343R, CXCR4E343k and CXCR4E343A, demonstrating that the negative charge is essential for mentioning physiological functions of CXCR4, otherwise, pathological events will occur, causing WHIM syndrome.Up to now, 105 cases of WHIM syndrome have been described all over the world, and almost all of them were caused by different mutations causing truncation of the C-terminus of CXCR4, losing phosphorylation sites, but only one missense mutation in CXCR4, and all potential phosphorylation are intact , 24–28. tization , consideWT Figs.  and 4c-fly Figs. , 4c-f. TE343K causing WHIM syndrome, expended our understanding of WHIM pathogenesis.We mutated the 343 site residue of glutamic acid (Glu/E) with to negatively charged residue asparitic acid (Asp/D), or positively charged residue lysine (Lys/K) or arginie (Arg/R), or non-charged residue alanine (Ala/A) to determine the signaling properties and functions of the CXCR4. Results from our studies have revealed that negative charge of 343 site is essential for physiologically functional CXCR4, charge-changing of residue at the 343 of receptor tailor dysregulates the down-stream G-protein signal events. This discovery is important for understanding the mechanisms of CXCR4The full-length CXCR4 (NM_003467.3) was amplified by PCR using primers: 5′-TCTAGAA TGGTGAGCAAGGGCGAGG-3′ and 5′-CTCGAGAATGCTGGAGTGAAAACTTTGAAG-3′, then was subcloned into a lentiviral plasmid pReceiver-Lv201. The mutant CXCR4 was generated by mutagenesis according the manufacturer’s instructions .HeLa and HEK293T cell lines were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences . Cells was maintained in MEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ ml penicillin and 100 μg/ml streptomycin  at 37 °C in a 5% CO2 incubator . The recAnti-ERK1/2 (1:1000), AKT (1:1000), JNK1/2/3 (1:1000) and p38 (1:1000) antibody were purchased from Cell Signaling Techonlogy . Anti-PI3K antibody (1:1000) was purchased from Bioworld Technology . Anti-phospho-Erk1/2 antibody (1:1000) was purchased from Boster Biological Technology . Anti-phospho-JNK1/2 antibody (1:1000) and anti-phospho-PI3K antibody (1:1000) were purchased from Immunoway . Anti-phospho-p38 antibody and anti-phospho-AKT antibody (1:1000) were purchased from Bioworld Technology . The anti-rabbit/mouse secondary antibody (1:1000) were obtained from Boster Biological Technology .The stably overexpressing HeLa cells were seeded into a 24-well plate and cultured in serum-free MEM overnight. When the cell density reached 80–90%, a scratch was introduced to the monolayer with 200 μL pipette tips. HeLa cells were washed for 2–3 times using PBS and incubated by MEM containing 5% FBS and 50 nM chemokine SDF-1  . The heaFor surface expression and internalization of the receptor analysis, stably transfected HeLa cells grown on six-plate were stimulated with 50 nM SDF-1 for 0 min or 0 min, 40 min at 37 °C. Reactions were terminated by addition of 4% PFA. Cells were stained with Mouse mAb IgG isotype control , anti-CXCR4 (4G10) primary antibody (Santa Cruz Biotechnology) and anti-mouse IgG(H + L) conjugate with Alexa Flour 647  and analyzed by using a four-color FACSCalibur flow cytometer equipped with CELLQUEST PRO software (BD LSRFortessa™ X-20) and analyzed using FlowJo software.4 Hela cells were seeded into the upper chambers  of Transwell (Millipore). Serum-free MEM medium with SDF-1 (50 nM) was added to the bottom chamber. After incubation for 5 h, cells remained on the upper membrane were removed with a cotton swab, while cells that had migrated through the membrane were fixed in formaldehyde, stained with crystal violet and imaged using Zeiss AxioScope A1 microscope, the cell number was quantified using ImageJ software [The HeLa cells were seeded into a 24-well plate and cultured in serum-free MEM overnight. 2 × 10software .Total RNA was extracted from cells using Trizol reagent  and reverse-transcribed into complementary DNA (cDNA)  using 500 ng RNA for qPCR according to the manufacturer’s instructions  The primers used in the qPCR were: IL-6 F: 5′-TGGATGAGCTGAACTGTACCC-3′, IL-6 R: 5′-GCTTGCCAAGGATT GTGAGT; TNF-α F: 5′-CGGAATTCGCCACCATGAGCACTGAAAGC-3′, TNF-α R: 5′-CGGA ATTCGCCACCATGAGCACTGAAAGC-3′; β-actin F: 5′-GATGGAAAGTGACCCGCA-3′, β-actin R: 5′-GAGGAAGACGCAGAGGTTTG-3′.The stably overexpressing HeLa cells were seeded into 6-well plates and cultured for 24 h, and maintained in 1 ml of serum-free MEM for 6 h at 37 °C before stimulation. After stimulation with SDF-1 (50 nM), cells were collected and washed three times with cold PBS, then total protein was extracted using RIPA lysis buffer . Proteins (30 μg) were separated via SDS-PAGE then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder in Tris-buffered saline (TBS) at room temperature for 1 h. Subsequently, the blots were incubated at 4 °C overnight with primary antibodies against phosphorylated p-ERK1/2, ERK1/2, p-JNK1/2/3, JNK1/2/3, p-p38, p38, p-PI3K, PI3K, p-AKT, AKT. The secondary antibodies were horseradish peroxidase (HRP)-linked anti-rabbit IgG, and HRP-linked anti-mouse IgG. The intensity of the protein bands was analyzed by the ImageJ software.The stable cell lines were grown and treated with SDF-1 (50 nM). Cells were fixed with 4% paraformaldehyde for 10 min at room temperature as described . ImmunosP-values via GraphPad Prism 7.0 software. The value P < 0.05 was considered as statistically significant.All experiments were replicated at least three times. The experiments results were presented as the mean ± standard deviation. A one-way analysis of variance (ANOVA) was utilized to calculate Additional file 1: Fig. S1 Ligand-induced internalization of wild type and mutant CXCR4. The mean levels of cell surface CXCR4 at the 40 min post-stimulation with SDF-1was examined by flow cytometry from 3 independent experiments. *p < 0 .05, **p <0 .01, ***p <0 .001 compared to the WT group.


The epidermal growth factor receptor (EGFR) inhibitors represent the first-line therapy regimen for non-small cell lung cancer (NSCLC). Most of these inhibitors target the ATP-site to stop the aggressive development of NSCLC. Stabilization of the ATP-binding on EGFR is difficult due to autophosphorylation of the EGFR domain. This leads to activation of nonintrinsic influence of the tumor microenvironment and expression of anti-apoptotic pathways and drug resistance.The NSCLC related literature search was carried out using online databases such as Scopus, Web of Sciences, PubMed, Protein Data Bank and UniPort for the last ten years and selected articles are referred for discussion in this review.To overcome the problem of mutations in NSCLC, the allosteric site of EGFR was targeted, which shows significant therapeutic outcome without causing resistance. Compounds like EAI001, EAI045 JBJ-04-125-02, DDC4002 and a series of small molecules with an affinity towards the EGFR allosteric site are reported and are under the investigational stage. These compounds are categorized under fourth-generation anti-NSCLC agents.Composition of this review highlights the advantage of inhibiting allosteric site in the EGFRTK receptor domains and presents a comparative analysis of the new fourth-generation anti-NSCLC agents to overcome the drug resistance. Epidermal growth factor receptor (EGFR) belongs to the family of protein kinase (PK) widely distributed throughout the human body. In the normal cell, EGFR is responsible for regulating the mTOR-serine/threonine-protein kinase pathway and maintenance of down-stream signaling pathway; which overall maintains the growth of hepatic cells . Any malIn the past few years, NSCLC has proven to be a serious economic burden, amounting to approximately US $2.6 billion being used for drug development and capitation with 8.6% cost inflation each year for the same . BesidesThe current treatment of NSCLC involves surgery, radiation, chemotherapy, and targeted therapy depending on the cancer stage. More than 40% of newly diagnosed lung cancers will be in stage IV, and cytotoxic combination chemotherapy is recommended as first-line therapy. The generally used chemotherapy includes platinum (cisplatin or carboplatin) in combination with paclitaxel, gemcitabine, docetaxel, vinorelbine, irinotecan, or pemetrexed. The drug paclitaxel also called as taxol, docetaxel a semi-synthetic taxol, vinorelbine a semi-synthetic vinca alkaloid act upon microtubule and polymerases or stabilizes it to inhibit the cell division. The drug gemcitabine inhibits the DNA synthesis required for cell divisions. The irinotecan a derivative of camptothecin inhibits topoisomerase I. The oreetrexed a antimetabolite antifolate agent used in combination for NSCLC. But, none of the single-drug regimens is significantly superior over a combination of two or more drugs . With thThe enzyme inhibition property of the EGFR-TK allosteric site targeting drugs results in a functional impact on the cell-signaling cascade . The novA literature search was conducted using online databases such as Scopus, Web of Sciences, PubMed, Protein Data Bank and Uniport for the last 10 years. The keywords used for the literature search were “lung cancer”, “NSCLC therapy”, “allosteric site EGFR”, “EGFR inhibitors”, “fourth-generation NSCLC drugs”, “anti-NSCLC agent”, “drawbacks EGFR inhibitor”. The literature search aimed to get all important information and research data, which can enhance the knowledge of allosteric site inhibition as a potential target in EGFR TK. The selected articles are referred for discussion in this review.0 and G1 phases of the cell cycle  of 17–32%, median progression-free survival (PFS) 3.1–5.5 months and median overall survival (OS) between 7.4 and 9.9 months. Platinum-based combination with cisplatin and carboplatin showed RR to be 25 and 28% respectively, and OS calculated to be 9.8 months and 8.2 months respectively . The maiDocetaxel is an anti-mitotic chemotherapeutic agent, used mainly for the treatment of ovarian, breast cancer, and NSCLC. Docetaxel reversibly binds to tubulin with high affinity in a 1:1 stoichiometric ratio. Pemetrexed is useful in combination with cisplatin for the treatment of patients with malignant pleural mesothelioma, which is either unresectable or not eligible candidates for curative surgery. This is due to the inhibition property of thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase of pemetrexed, which makes it antifolate agent . The comThe first-generation EGFR drugs, namely gefitinib and erlotinib, are approved for the treatment of NSCLC in 2003 and 2004 by US-FDA. These drugs are EGFR-ATP binding inhibitors. Their efficacy failed due to secondary mutation. Threonine-790 (T790M) is tagged to be the “gatekeeper residue” for the inhibition of activation of the ATP site in EGFR . This diThe first generation gefitinib use was restricted in 2005 and later withdrawn in 2012 by US-FDA on the basis of negative results in phase-III trials. But there are opinions that they can still have their role in spite of third-generation drugs . To overThe EGFR secondary mutation or T790M mutation is the major reason for the resistance development in the first and second-generation drugs . OsimertZhang et al. 2015) studied the combination of cetuximab with other anti-cancer drugs like docetaxel and pemetrexed individually. Even though the patient did not show complete response to the combination therapy, the objective response rate (ORR) was 11.8%, compared to the disease control rate (DCR) 50% and disease progression rate (DPR) 50%. This therapy regimen identified to be as salvage therapy when all other drugs fail to respond. One of the major drawbacks was that the therapy showed the response only to the EGFR-TK pathway and had a delayed response of over 6 months l-tyrosylThe kinase structure has five major regions, namely, N-lobe, C-lobe, ATP site, hinge region and the allosteric site Fig. . N-lobe Drugs developed so far for lung cancer are protein kinase antagonist, which are a steady-state competitive inhibitor . The ATPThe distinguishing feature of EGFR is the prolonged activation of auto-phosphorylation. Major ligands that interact with TK and lead to dimerization of the TK complex are an epidermal growth factor (EGF), tumor necrosis factor (TNF), transforming growth factor α (TGF-α), epiregulin, and amphiregulin. The EGFR family attains consecutive kinase activation even in the absence of phosphorylation and diametric state attenuation. Hence, the receptor remains inactive even in the absence of ligand interaction or activation factor. Once it is activated, phosphorylation takes place without the involvement of ligand. This phosphorylation leads to prolonged dimerization of the TK-domain. The currently available drugs interact with the receptor partially, which further leads to steric hindrance between the drug and receptor, leading to a decrease in therapeutic efficacy. This prolonged conformational change in the TK receptor affects the drug interactions with the receptor. The drug binds weakly to the receptor, which further decreases the overall therapeutic efficacy. Hence, to achieve the therapeutic index, the patients are subjected to a high dose regimen over time; which subsequently leads to the development of resistance and hepatotoxicity . Thus thThe first, second and third-generation drugs have failed to produce therapeutic benefit due to EGFR mutations. Targeting the allosteric site has gained attention for the therapeutic relief for many indications . The vasA TK receptor has three binding sites, namely, ATP competitive site, inactive site and allosteric site. The most of the currently available drugs act on ATP binding site and there are no drugs which act on inactive site. Inactive site of the receptor is the region where the protein does not interact with ligand or drugs. This region is not directly involved with the pharmacotherapeutic response. The binding of effector to the allosteric site on the protein leads to conformational changes at the active or binding site, which regulates protein activity . AllosteThus, the novel properties and characteristics of the allosteric site help to overcome the problem of “undruggable” proteins and reduces the chances of developing side effects and adverse effects of the therapy. If such proteins have transient allosteric pockets, then the proteins can be explored for its direct or indirect activity. Targeting the allosteric site can lead to collective therapy development and helps explore the pharmacology of such proteins. For this reason, the allosteric site retains the potential to become the novel target for the development of anti-NSCLC agent .When allosteric site inhibitors bind with the ATP site but do not interact with the hinge region, this leads to blockade in kinase activity without displacing ATP Fig. . FurtherJia et al.  and Zhaopara-fluorophenol moiety, which interacts with Met790 and Phe856 side-chain. The compounds, EAI001 and EAI045, showed inhibitory activity due to hydrophobic interaction with amino acid Ile759, Leu747, Leu788, Leu777 and Met766. These compounds are polar due to the interaction of its nitrogen atom in the amide moiety with Asp 855 in the allosteric site of EGFR. Compound EAI001 has different confirmation as compared to EAI045 due to its interaction with gene T790M/V948R and ANP, which hinders the free movement of the Met790 side chain  of which 38% helix and 14% is β-sheets. EAI045 has three residues, namely ANP A1102, which is ATP moiety; 57N 1103, which is an inhibitory ligand and MGA 1101, which is magnesium ion moiety . The whopara-fluorophenyl in place of phenyl group at the ortho position thiazole ring of EAI001  is observed to be 62–71%, and progression-free survival (PFS) was 9.9–12.5, which is more efficacious than chemotherapy regimen  diazepine-11-one scaffold. It comprises of a seven-membered diazepinone ring, which is packed inward the α-C-helix. This compound has been reported to inhibit the L858R/T790M and L858R/T790M/C797S EGFR mutation and have IC50 of ~ 0 nM. The compound DDC4002, when interacting with EGFR allosteric site, leads to the extension of N-lobe leading to benzyl substitution between AMP and PNP, side-chain of K795, L788 and T790M, the gatekeeper-mutation in cells. This ultimately leads to inhibition of cell proliferation and inhibition of caspase-7 pathway. The reports also suggest that DDC4002 is more efficacious than compound EAI045. DDC4002 showed EGFR biochemical activity against L858, therefore, DDC4002 have the potential to become an anti-NSCLC agent and can solve the drug resistance development problem effectively [Compound DDC4002, (PDB i.d.: 6PID), have a 5,10-dihydro-11ectively .50 ~ 14 µM. Among these compounds, the compound 8 reported the least IC50 or highest EGFR inhibitory activity (Table R)-enantiomer of eight hydrogen bond to Lys745 side chain. The nitrogen molecule in indole showed hydrogen bond formation with the Phe856 carbonyl group in the allosteric site of EGFR. The nitrophenylalanine of the group showed hydrogen bond interaction with Asp855 of the receptor [S)-enantiomer conformation of these compounds with the carbonyl group of Asp 855 [Caporuscio et al. , have rety Table . This is Asp 855 .NSCLC is the leading cause of death among all oncology cases. EGFR is a potential target for developing drugs in NSCLC . The eff


CaMKII activity depends on the balance between activating and inhibitory autophosphorylation . Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.The many variants of human Ca The fou2+/calmodulin (Ca2+/CaM) to the regulatory segment , with relatively little inhibitory phosphorylation on Thr 305/306. This is consistent with expectation . Unexpec2+/CaM and ATP, we found that the dephosphorylation by phosphatases of activating phosphorylation at Thr 286 is much slower than dephosphorylation of inhibitory phosphorylation at Thr 305/306, which is fast. The protection of activating phosphorylation and the rapid reversal of inhibitory phosphorylation by phosphatases allows CaMKII to act as an integrator of Ca2+ pulses in cells. CaMKII-α and CaMKII-β can assemble to form mixed holoenzymes, with the balance between activating and inhibitory autophosphorylation determined by the ratio of the two isoforms. Thus, the calcium responsiveness of CaMKII holoenzymes can be tuned by varying the relative levels of the two isoforms.When CaMKII is inactivated after activation, by removing CaWe designed a single-molecule assay to measure autophosphorylation of CaMKII, relying on immediate capture of the holoenzyme after lysis of mammalian cells overexpressing CaMKII  . This prWe quantify the extent of phosphorylation in two ways. First, we measure the fraction of CaMKII holoenzymes that show detectable phosphorylation at Thr 286, regardless of the extent of phosphorylation, by monitoring the co-localization of green and red spots . To quan2+/CaM and ATP over the glass coverslip to which the captured holoenzymes are tethered , resulted in the detection of increasing levels of phosphorylation at Thr 286 . We activated CaMKII-α by flowing Catethered . Increas Thr 286 . The EC5Ca2+/CaM .A limitation of this assay is that the integrated intensity values cannot be used to determine the absolute number of phosphate groups within each holoenzyme, because we do not know whether every phosphate group necessarily has an antibody bound to it when the maximum signal is obtained. Detection of phosphorylation may be less than complete because of steric interference between antibodies, or interference with the glass support. Due to this limitation, we use the values of the integrated antibody fluorescence intensities to provide a relative, rather than an absolute, measure of the extent of phosphorylation.2+/CaM (5 μM) and 500 μM ATP.We used the single-molecule assay to compare the levels of phosphorylation at the activating (Thr 286) and inhibitory (Thr 305/306) sites in CaMKII-α and CaMKII-β. We expressed CaMKII-α and CaMKII-β separately in HEK 293T cells and, in parallel experiments, captured the CaMKII isoforms on glass, followed by activation with high concentrations of CaThese experiments reveal clear differences in the extent of autophosphorylation at the inhibitory sites in CaMKII-α and CaMKII-β. Whereas 95% of the CaMKII-β holoenzymes show detectable phosphorylation at Thr 305/306, only 30% of the CaMKII-α holoenzymes do so -right. T2+/CaM. This was followed by the addition of ATP to the flow cell. ATP treatment of pre-activated CaMKII led to robust phosphorylation of CaMKII-α on Thr 305/306, at levels comparable to that for CaMKII-β  and CaMKII-δ (31-residue kinase-hub linker). We created surrogates for the two isoforms using CaMKII-α, denoted as CaMKII-γ* and CaMKII-δ*. As for CaMKII-β*, the kinase-hub linker in CaMKII-α is replaced with those from the γ and δ isoforms, respectively, in these surrogates. The results for CaMKII-γ*, with the long linker, resemble those for CaMKII-β/CaMKII-β* . LikewisThis conclusion was further reinforced by studying a naturally occurring splice-variant of CaMKII-β (CaMKII-β’E) that has a 55-residue linker, created by deletion of 155 residues. We also designed a construct where this truncated linker from CaMKII-β’E replaced the linker in CaMKII-α (CaMKII-β’E*). For both these constructs, we see a decrease in inhibitory phosphorylation and a small increase in activating phosphorylation when compared to CaMKII-β . As an a2+/calmodulin.CaMKII-α and CaMKII-β can assemble into mixed holoenzymes containing both isoforms . We co-tTo understand how changes in the length of the kinase-hub linker can alter the balance of autophosphorylation, we analyzed a simple kinetic model for CaMKII activation in which the holoenzyme contains only two kinase domains (see Appendix). A kinetic model for the activation of a dodecameric CaMKII holoenzyme requires the specification of an extremely large number of intermediate states, and we have not pursued this. In our simple kinetic model, we assume that Thr 286 can only be phosphorylated in trans, because it is located too far from the active site of the kinase . We assutrans-phosphorylation of either Thr 286 or Thr 305/306, the kinase acting as the enzyme has to have Ca2+/CaM bound to it, otherwise the active site of the enzyme will be blocked. For trans-phosphorylation of Thr 286, the kinase acting as the substrate must also have Ca2+/CaM bound to it, otherwise the Thr 286 is not accessible for phosphorylation , phosphorylation at this site is suppressed . Our results show that when the rate of trapping . Since Cppressed . The sitrylation . In this2+ levels. The cells were then lysed and CaMKII holoenzymes were captured and assayed as before, without any further treatment.We tested whether the balance between activating and inhibitory autophosphorylation is maintained when the enzyme is activated in cells. We overexpressed CaMKII-α or CaMKII-β* in HEK 293T cells, along with calmodulin, followed by ionomycin treatment to increase intracellular CaPhosphorylation at Thr 286 increases upon treatment with 10 μM ionomycin for both CaMKII-α and CaMKII-β*, as expected . CaMKII-2+/CaM and ATP on glass, while including increasing amounts of λ-phosphatase, a potent non-specific phosphatase , followed by the addition of saturating amounts of λ-phosphatase. Samples were pulled down from this reaction mixture at defined time points, and the phosphorylation at Thr 286 and Thr 305/306 was measured using the single-molecule assay. These experiments also showed slower rates of dephosphorylation at Thr 286 when compared to Thr 305/306  . CaMKII- 305/306 .2+/CaM to phosphorylated CaMKII enhances the rate of dephosphorylation at Thr 286 by about 4-fold for both CaMKII-α and CaMKII-β* .The observed resistance of pThr 286 towards rapid dephosphorylation suggests that this site may be less accessible to phosphatases compared to pThr 305/306. Unexpectedly, we found that the addition of CaaMKII-β* . When Ca2+/CaM has no effect on the rate of dephosphorylation of pThr 305/306, indicating that Ca2+/CaM does not increase the activity of the phosphatase. These data suggest that, in the absence of Ca2+/CaM, the calmodulin-binding element of CaMKII plays a role in sequestering pThr 286 from phosphatases. The mechanism underlying this protection remains to be understood.The addition of Ca2+/CaM-independent activity after a calcium pulse has subsided. CaMKII subunits that are phosphorylated on Thr 286 have increased affinity for Ca2+/CaM, which can allow a subthreshold Ca2+-pulse to cooperatively spread Thr 286 phosphorylation within the primed holoenzyme , the extent of phosphorylation for holoenzymes that show detectable pThr 286 is restored to the levels seen for fully activated CaMKII-α . Thus, in contrast to CaMKII-α holoenzymes, CaMKII-β holoenzymes tend to become inactivated when subjected to subthreshold levels of Ca2+/CaM, rather than undergoing priming for further activation.A similar experiment using CaMKII-β* yields very different results. Here, phosphatase treatment results in substantial reduction in pThr 286 levels. Subsequent treatment with 25 nM CaCaMKII is unusual among protein kinases because it is assembled into a large holoenzyme with twelve to fourteen subunits. Analysis of the phosphorylation status of the enzyme using bulk assays, such as Western blots, has been challenging due to difficulties in purifying the holoenzymes, particularly for isoforms with longer kinase-hub linkers. In addition, bulk measurements do not provide a window into the extent of phosphorylation per holoenzyme. To overcome these limitations, we designed a single-molecule TIRF microscopy assay that relies on the capture from mammalian cell lysates of CaMKII holoenzymes tagged with fluorescent proteins. Immobilization of the captured holoenzymes in a flow-cell apparatus allows activation and dephosphorylation of CaMKII to be carried out readily, followed by measurement of the phosphorylation status using fluorescently labeled site-specific antibodies. Using this assay, we discovered that CaMKII-α and CaMKII-β, the two major isoforms in the brain, differ in the balance of activating and inhibitory phosphorylation. CaMKII-α, with a shorter linker, readily acquires activating phosphorylation, while CaMKII-β, with a longer linker, is biased towards inhibitory phosphorylation. We also tested the effects on autophosphorylation of the kinase-hub linkers from CaMKII-γ (110 residues) and CaMKII-δ (31 residues). The shorter CaMKII-δ linker behaves like that of CaMKII-α in terms of autophosphorylation outcome. The outcome for the longer CaMKII-γ linker is closer to that seen with the CaMKII-β linker.trans-autophosphorylation within a holoenzyme, rather than phosphorylation between different holoenzymes , which are connected to cAMP-dependent protein kinase (PKA) by long flexible linkers. It has been suggested that the length and flexibility of the AKAP linker determines the spatial range over which PKA can phosphorylate its substrates . Since C2+/CaM, suggesting that the calmodulin-binding element of CaMKII adopts a conformation that protects pThr 286 in the absence of Ca2+/CaM. The presence of such a conformational change may explain the apparent discrepancy between our results and studies reporting that pThr 286 undergoes very rapid dephosphorylation in neurons  after modifying the vector backbone to remove the SNAP-tag. EYFP-CaM was a gift from Emanuel Strehler (Addgene plasmid # 47603). The EYFP tag was removed from this vector to generate the mammalian expression construct for calmodulin. All constructs with large domain insertions and deletions were made using standard protocols for Gibson assembly . All point mutants used were generated using the standard Quikchange protocols .Human CaMKII-α (Uniprot_ID: Q9UQM7) and human CaMKII-β (Uniprot_ID: Q13554) were cloned into the pEGFP-C1 vector backbone , after modifying the vector to contain a biotinylation sequence  followed by a linker (GASGASGASGAS) at the N-terminus of mEGFP. CaMKII-α or CaMKII-β was cloned at the C-terminus of mEGFP, with a linker sequence (PreScission protease site: LEVLFQGP) separating the mEGFP tag from the coding sequence of CaMKII-α/CaMKII-β . These constructs were then used as a template to produce the other CaMKII-variants. The splice-variant of CaMKII-β (CaMKII-β’E) was produced by deleting 155 residues the β-isoform (Uniprot_ID: Q13554-3). CaMKII-β*, CaMKII-γ*, CaMKII-δ*, and CaMKII-β’E* were produced by replacing the linker in CaMKII-α with that from CaMKII-β (Uniprot_ID: Q13554), CaMKII-γ (Uniprot_ID: Q13555), CaMKII-δ (Uniprot_ID: Q13557), and CaMKII-β’E, respectively . mCherry2. Transient transfection of CaMKII variants were done using the standard calcium phosphate protocol (2O (10X), 250 mM CaCl2 was added and the mixture was allowed to sit for 15 min at room temperature. Following this incubation, 2X HBS buffer  was added to it dropwise and mixed thoroughly by reverse pipetting. This mixture was then added to the HEK 293T cells and the cells were allowed to express the protein for 18–20 hours before harvesting.HEK 293T cells were obtained from the UC Berkeley cell culture facility (authenticated using STR profiling and tested negative for mycoplasma). These cells were grown in Dulbecco's Modified Eagle Medium + GlutaMaX  that is supplemented with 10% FBS , Antibiotic-Antimycotic  at 100X dilution and 20 mM HEPES buffer and maintained at 37°C under 5% COprotocol . BrieflyAll single-molecule experiments were performed in flow chambers  that were assembled with functionalized glass substrates . The glass substrates were first cleaned using 2% Hellmanex III solution  for 30 min, followed by a 30 min sonication in 1:1 mixture (vol/vol) of isopropanol:water. The glass substrates were then dried with nitrogen and cleaned for another 5 min in a plasma cleaner . These cleaned glass substrates were used to assemble the flow chambers immediately after plasma cleaning. After assembly, the glass substrates were treated with a mixture of Poly-L-lysine PEG and PEG-Biotin  for 30 min . The glass substrates were then washed with 2 mL of Dulbecco’s phosphate-buffered saline . Streptavidin  was added to these glass substrates at a final concentration of 0.1 mg/mL and incubated for 30 min. Following incubation, excess streptavidin was washed away using 2 mL of DPBS and these assembled flow chambers with streptavidin-coated glass substrates were used for all our single-molecule experiments.E. coli biotin ligase, BirA, with the CaMKII variants bearing an Avitag, results in the biotinylation of CaMKII in HEK 293T cells. After harvesting, the cells were lysed in a lysis buffer containing 25 mM Tris at pH 8, 150 mM KCl, 1.5 mM TCEP-HCl, 1% protease inhibitor cocktail , 0.5% phosphatase inhibitor cocktail 2  and 3 , 50 mM NaF, 15 μg/ml benzamidine, 0.1 mM phenylmethanesulfonyl fluoride and 1% NP-40 (Thermo Fisher). The cell lysate was then diluted 100–200 times in DPBS and 100 μL of this diluted cell lysate was added to a well in the flow chamber for 1 min, before washing it out with 1 mL of DPBS. During this incubation, the biotinylated mEGFP-CaMKII variants were immobilized on the surface of the functionalized glass substrates, owing to the streptavidin-biotin interaction. A buffer exchange was then done in the flow chamber to replace the DPBS with the gel filtration buffer .CaMKII variants were allowed to express for 18–20 hours before they were harvested. The co-expression of the 2, 10 mM MgCl2, 500 μM ATP in the gel filtration buffer and the CaM concentration varies between 0.02–5 μM CaM  depending on the experiment) into the flow chambers for 60 min. Following this incubation, the activation buffer was washed out using 2 mL of the gel filtration buffer. All experiments have been replicated at least 3–5 times using samples prepared over different days.The glass-immobilized CaMKII holoenzymes were activated by flowing in an activation buffer  for 10–15 min, activating CaMKII within the cell. Unactivated samples were obtained from cells treated with HBSS buffer alone for the same amount of time. The cells were then lysed, and activated or unactivated CaMKII was pulled down on functionalized glass substrates without further treatment. For experiments that involved incubation with phosphatase inhibitors, 250 nM cyclosporin A  and 250 nM okadaic acid  were added to the cells for 60 min prior to the activation of CaMKII. The same procedure as above was then followed to activate CaMKII in cells and pull down activated or unactivated CaMKII directly from the cell lysates.HEK 293T cells overexpressing CaMKII and CaM were washed with HBSS buffer  plus 25 mM HEPES and incubated at room temperature for 5–10 min. The cells were then treated with 10 μM ionomycin  and 3 mM CaClThe autophosphorylation status of the CaMKII holoenzyme at the activating (Thr 286) and the inhibitory (Thr 305/306) sites was estimated using phosphospecific antibodies for pThr 286 (Abcam: ab171095) and pThr 305/306 (PhosphoSolutions: p1005-306). The immobilized, activated and unactivated CaMKII holoenzyme was incubated with a mixture of these two phosphospecific primary antibodies (in a 1:1 ratio using a 1:500 dilution in 5% (w/v) BSA) for 45 min. Subsequently, excess primary antibodies were washed out with 3 mL DPBS. This was followed by a 30 min incubation with a 1:1 mix of Alexa-labeled secondary antibodies (1:1000 dilution in 5% BSA), that are complementary to the primary antibodies used. Anti-mouse secondary antibody labeled with Alexa-594  was used for pThr 286-specific primary antibody; anti-rabbit secondary antibody labeled with Alexa-647  was used for the pThr 305/306-specific primary antibody. After incubation with the secondary antibody, the flow chambers were washed again using 3 mL of DPBS and the samples were imaged using Total Internal Reflection Fluorescence (TIRF) microscopy.286 is mutated to A286 and the epitope for the Thr 305/306 primary antibody is changed from T305T306MLATRNFS to A305V306I) in CaMKII-α . After 45 min of incubation, the flow chambers were washed with 2 mL DPBS to remove the λ-phosphatase and the activation buffer. The phosphorylation of Thr 286 and Thr 305/306 was then examined using the immunofluorescence assay described above. No further reduction in phosphorylation was observed upon adding more than 400 units of λ-phosphatase and this is considered as a saturating amount of phosphatase.To test how the presence of phosphatase affects the phosphorylation status of CaMKII when the kinase is active, increasing amounts of λ-phosphatase  and 1 mM MnCl2 and 400–800 units of λ-phosphatase or 800 units of PP1α was added to the flow chambers displaying activated CaMKII, after the activation buffer had been washed out. 1 unit of PP1α is defined as the amount of enzyme that releases 1 nmol phosphate per minute from the phosphorylated substrate DiFMUP  (EMD Millipore). The dephosphorylation reactions were carried out for defined periods of time , following which the phosphatase buffer was washed away with 2 mL DPBS and the autophosphorylation states were examined using the immunofluorescence assay described above.To test the sensitivity of the two autophosphorylation sites to phosphatases in the absence of kinase activity, activated CaMKII was treated with two different phosphatases, λ-phosphatase (New England Biolabs) and PP1α . A phosphatase buffer that contains 1 mM MnCl2+/CaM and 5 mM Tris-buffered TCEP (Sigma-Aldrich). After activation for 45 min, 100 μM staurosporine  was added to this reaction mix to inhibit the kinase activity. After inhibiting CaMKII for 10 min, saturating amounts of λ-phosphatase (400 units) was added to the solution. Samples were pulled down after 3 or 15 min of incubation and the phosphorylation status on Thr 286 and Thr 305/306 was measured. We verified that 100 μM staurosporine was efficient in switching off kinase activity by activating CaMKII-α/β* in the presence of the inhibitor. This treatment completely abolished any autophosphorylation of Thr 286 or Thr 305/306 (The HEK 293T cell lysate was diluted by 1:100–200. CaMKII was activated in this diluted lysate (in solution) by adding an activation buffer containing 5 μM Ca 305/306 .2 for 3–5 min, followed by washing away of the phosphatase buffer with 2 mL of DPBS. This sample was then incubated with an activation buffer (see details above) containing subthreshold concentrations of 25 nM Ca2+/CaM for 30 min. The activation buffer was aspirated out with 2 mL DPBS and the phosphorylation status at the activating and inhibitory sites were measured.Activated samples of glass-immobilized CaMKII were treated with saturating amounts of λ-phosphatase and 1 mM MnClSingle-particle total internal reflection fluorescence images were acquired on a Nikon Eclipse Ti-inverted microscope equipped with a Nikon 100 × 1.49 numerical aperture oil-immersion TIRF objective, a TIRF illuminator, a Perfect Focus system, and a motorized stage. Images were recorded using an Andor iXon electron-multiplying charge-coupled device camera. The sample was illuminated using the LU-N4 laser unit  with solid state lasers for the 488 nm, 561 nm and 640 nm channels. Lasers were controlled using a built-in acousto-optic tunable filter (AOTF). The 405/488/561/638 nm Quad TIRF filter set  was used along with supplementary emission filters of 525/50 m, 600/50 m, 700/75 m for 488 nm, 561 nm, 640 nm channel, respectively. 3-color image acquisition was performed by computer-controlled change of illumination and filter sets at 42 different positions from an initial reference frame, so as to capture multiple non-overlapping images. Image acquisition was done using the Nikon NIS-Elements software. mEGFP-CaMKII, Alexa-594-labeled anti-Thr 286 antibody, and Alexa-647-labeled anti-Thr 305/306 antibody were imaged by illuminating 488 nm laser set to 5.2 mW, 561 nm laser set to 6.9 mW, and 640 nm laser set to 7.8 mW, respectively. The laser power was measured with the field aperture fully opened. Images were acquired using an exposure time of 80 milliseconds for 488 nm and 561 nm, and an exposure time of 100 milliseconds for 640 nm. The only exception was for acquiring mCherry single-molecule images, where an exposure of 200 milliseconds was used. Epifluorescence images were also acquired using a Nikon Eclipse Ti-inverted microscope with a Nikon 20x objective and an Andor iXon electron-multiplying charge-coupled device camera. A mercury arc lamp  was used for epifluorescence illumination at 488 and 561 nm.2 were excluded due to heterogeneous TIRF illumination. No further filtering processes was applied in TrackMate.Individual single particles in all three channels were detected and localized using the single particle tracking plugin TrackMate in ImageJ . The parIntensity distribution and fraction of holoenzymes showing any detectable phosphorylation were analyzed using custom in-house software written in MATLAB (these programs are provided as a source code file). For analyzing the fraction of holoenzymes that show any detectable phosphorylation at a given phosphosite, the XY-coordinates for each particle were extracted from the TrackMate data. For each mEGFP-CaMKII, distances to all the antibody particles were computed, and colocalization was identified if the inter-particle distance was below two pixels. The fraction of CaMKII holoenzymes  with detectable phosphorylation was computed as a ratio of the number of CaMKII colocalized with antibody to the total number of CaMKII holoenzymes. In a typical case, false-positive colocalization due to coincidentally neighbored particles was less than 0.5%, as determined from analyzing multiple, unrelated sets of images with comparable particle density. The intensity values acquired from TrackMate statistics data for the pThr 286 and pThr 305/306 antibody-channels were used to calculate an intensity histogram, for the colocalized  and the non-colocalized spots . This results in scaling of the area under the histogram for the antibody intensity by a factor that takes into account the fraction of holoenzymes that show no detectable phosphorylation. A bin width of 500 was used for computing the histogram. After calculating the distribution, we plotted the intensity histogram for only the colocalized spots and an intensity cutoff of about 15000 arbitrary units.For analysis of samples where mEGFP-CaMKII-α and mCherry-CaMKII-β* were co-transfected , we first identified the heterooligomeric holoenzyme population  and then calculated what fraction of these heterooligomers showed detectable inhibitory phosphorylation. An intensity histogram was also plotted for the pThr 305/306 signal for these holoenzymes, as described above. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The studies provide new insights into the mechanisms of activation and inactivation of CaMKII in response to elevated calcium. They highlight a role for the length of the linker between the catalytic domain and the hub of CaMKII in determining the extent to which an activating versus an inhibitory autophosphorylation occurs in response to calcium, and explains why the kinetics of activation and inactivation of the CaMKII-α and CaMKII-β isoforms are different. Overall, this paper provides a significant advance in our understanding of the regulation of this key protein kinase.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Tony Hunter as the Reviewing Editor and Jonathan Cooper as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: John D Scott (Reviewer #1); Stefan Knapp (Reviewer #2).Thank you for submitting your article "Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation" for consideration by eLife.Our decision has been reached after consultation between the three reviewers. Based on these discussions, we are pleased to inform you that we will offer you the opportunity of submitting a revised version of your paper to eLife, and they may stimulate more research focusing on the role of the CaMKII-β isoform in more relevant cell types.The new insights you have obtained through your biochemical, single molecule studies of the mechanisms of activation and inactivation of CaMKII in response to elevated calcium, in which the length of the linker between the central hub and the catalytic domain dictates whether autophosphorylation is primarily at the T286 activating site or at the T305/306 inhibitory sites, provide a significant advance in our understanding of the regulation of this key enzyme. Your studies have led to the formulation of an intricate model for the activation and inactivation of CaMKII's that may prove challenging for some readers to fully appreciate, but the in vitro data are of sufficient interesting for publication in 2+/CaM-activated autophosphorylation events, they indicate that some further experiments with additional linker lengths would strengthen your conclusions. Currently, you only tested the natural ~30 and ~200 residues. However, it would be of mechanistic interest to know whether the 30 residue linker (in CaMKII-α) is the optimal length for Thr286 trans-autophosphorylation, or whether the optimal length is significantly shorter or longer than this. Likewise, what is the minimal linker length for inhibitory autophosphorylation? Testing additional, intermediate linker lengths would enable you to address this – perhaps natural CaMKII isoform linkers of different lengths exist that could be tested. Also, for completeness sake, it would be ideal if you tested the reciprocal CaMKII-α/β chimera, i.e., CaMKII-α*, in which the CaMKII-α linker is swapped into CaMKII-β.The main experimental issue to be addressed is as follows. While the reviewers agree that the most interesting outcome of your analysis is the influence of the length of the catalytic domain-hub linker on the nature of the CaAnother concern is that the model still represents a rather artificial system of ectopically expressed fusion proteins. In particular, the λ phosphatase used here is known to have very broad specificity, suggesting that more relevant and selective Ser/Thr phosphatases that are known to interact and regulate CaMKII, such as PP1 and PP2A, may produce different dephosphorylation kinetics. λ phosphatase is a rather small, highly active protein of ~24 kDa, whereas the eukaryotic cell PPI and PP2A phosphatase holoenzyme complexes are significantly larger, and it is certainly possible that the kinetics of CaMKII dephosphorylation by these enzymes might be significantly slower, perhaps because of steric access issues.Reviewer #1:As stated by Bhattancharyya et al. CaMKII is unusual among protein kinases because it is assembled into a large holoenzyme with twelve to fourteen subunits. Analysis of the phosphorylation status of the enzyme using bulk assays, such as Western blots, has been challenging due to difficulties in purifying the holoenzymes, particularly for isoforms with longer kinase-hub linkers. To circumvent this problem the authors designed an elegant designed a single-molecule TIRF microscopy assay that relies on the capture from mammalian cell lysates of CaMKII holoenzymes tagged with fluorescent proteins. Using this approach they conclude that CaMKII-a and CaMKII-b, the two major isoforms in the brain, differ in the balance of activating and inhibitory phosphorylation. This can be explained in part by the differences in the length of a flexible linker that differs between the CaMKII-a and CaMKII-b isoforms.Overall, this is a well written and detailed study. The rigorous biochemical approach of the Kuriyan lab is balanced with the power of single molecule fluorescence approaches that have been pioneered by the Grove lab. This has led to the formulation of an intricate model for the activation and inactivation of CaMKII's that may prove challenging for some readers to fully appreciate. I only have a few minor comments.1) I wonder if there is merit in incorporating Figure S3 into the body of the text. I recognize that this is already a vast study of nine full figures, but the data on dephosphorylation in a cellular context is a nice addition to the study.2) Figure 5 is necessary but quite complicated. I wonder if there is any way to simplify this section.3) Experiments with small molecule inhibitors such as staurosporine are appropriate, but I wonder if there would be benefit from including work with more established CaMKII antagonists such as KN62.Reviewer #2:Bhattacharyya et al. studied the autophosphorylation kinetics of the two main Ca/calmodulin-dependent protein kinase II (CaMKII) isozymes expressed in brain: CaMKII-α and CaMKII-β. To do this, the authors developed a single-molecule assay to measure the phosphorylation status of CaMKII using expression constructs of CaMKII isoforms expressed with a N-terminal fluorescent proteins (mEGFP) fused to a biotin tag for easy capturer on coated glass plates and subsequent visualization, by total internal reflection fluorescence (TIRF) microscopy. Phosphorylation states were detected by phosphorylation site specific antibodies recognizing the activating phosphorylation site Thr286 as well as the two inhibitory sites Thr305/306.2+ influx. Kinases of this family form dodecameric or tetradecameric holoenzymes that may contain also contain heteroligomeric assemblies of different isoforms that share high sequence homology. The oligomeric state facilitates auto-phosphorylation within the same oligomer allowing for rapid amplification of transient Ca signals. The authors make the intriguing discovery that the linker region between the oligomerization domain and the kinase domain which is much longer in the β isoform modulates autophosphorylation behaviour: CaMKII-α which has a 31-residue linker quickly autophosphorylated on the activating site (Thr286) with relatively little inhibitory phosphorylation on Thr305/306. In contrast, CaMKII-β, which has a longer ~200-residue linker preferentially autophosphorylated at the inhibitory sites Thr305/306 with less activating phosphorylation on the activating site Thr286. A mutant of CaMKII-α that contains the β linker confirmed that the difference in autophosphorylation is indeed a result of the linker region and not due to differences in the catalytic or oligomerization domains. Thus, CaMKII activity in cell can potentially regulated by different ration of α and β isoforms, generating heterooligomers with variable response to activating Ca/calmodulin signals. In addition, de-phosphorylation was studied using non-specific l-phosphatase showing again linker dependent differences.CaMKII constitute a family of enzymes that play a key role in learning and memory by mediating signalling after CaThe reported activation and inactivation data of this key enzyme system are certainly interesting. Experimental data are sound and have been interpreted with care. My only concern is, that the model still represents a very artificial system of ectopically expressed fusion proteins. In particular, the phosphatase used is known to have very broad specificity suggesting that more relevant and selective Ser/Thr phosphatases that are known to interact and regulate CaMKII such as PP1 and PP2a, may produce different dephosphorylation kinetics. However, I think that the in vitro data are however sufficiently interesting for publication in eLife when limitations are more clearly highlighted and they may stimulate more research focussing on the role of the β isoform in more relevant cell types.Specifically:a) The notion that α and β ratios regulate activity could be supported by (available) expression data in different cell types. Such data would at least demonstrate that the proposed regulatory mechanism is indeed observed.2+ pulses" suggests that signalling events have been studied downstream of Ca2+ pulses – this is not the case as only dephosphorylation kinetics by l-phosphatase has been experimentally assessed. The authors should also discuss that CaMKII activation in neurons is extremely transient (approximately 1 min duration). This is probably not within the time resolution of the current experimental setup.b) The heading "The balance between inhibitory and activating autophosphorylation is maintained upon activation of CaMKII in cells" is misleading. The experiments are carried out in lysates using ectopically expressed protein in HEK293T cells. Similarly, the heading "Slower dephosphorylation at the activating site primes CaMKII for activation by incoming CaReviewer #3:This biophysical study explores the differences in the autophosphorylation behavior of CaMKII-α and CaMKII-β. These two isoforms differ mainly in the length of the flexible linker between the N-terminal kinase domain and the C-terminal hub domain: ~30 residues for CaMKII-α and ~200 residues for -β. The three autophosphorylation sites examined were Thr286 and Thr305/306, just C-terminal to the kinase domain. It had been shown previously that Thr286 is autophosphorylated in trans (but within the oligomeric CaMKII structure) and activates CaMKII kinase activity, and that Thr305/306 can be autophosphorylated in cis or in trans and is inhibitory. Using single-molecule TIRF microscopy, the authors' main finding is that autophosphorylation of Thr286 is dependent on the length of the kinase-hub linker, with a shorter linker (as in CaMKII-α) facilitating Thr286 autophosphorylation (in trans). They also explored the susceptibility of these phosphorylation sites to phosphatase activity.eLife. Given the authors' main finding that the kinase-hub linker length plays a crucial role in the activating/inhibitory phosphorylation ratio, one would think that they would have explored this feature more exhaustively, for example, by testing intermediate linker lengths . Is 30 residues (in CaMKII-α) the optimal length for Thr286 trans-autophosphorylation, or is the optimal length shorter or longer? Also, for completeness sake, the authors should test the reciprocal CaMKII-α/β chimera, i.e., CaMKII-α*, in which the CaMKII-α linker is swapped into CaMKII-β.Although these results will be of interest to those in the CaMKII field, it is debatable whether the study, in its current form, is of high enough significance to merit publication in  2+/CaM-activated autophosphorylation events, they indicate that some further experiments with additional linker lengths would strengthen your conclusions. Currently, you only tested the natural ~30 and ~200 residues. However, it would be of mechanistic interest to know whether the 30 residue linker (in CaMKII-α) is the optimal length for Thr286 trans-autophosphorylation, or whether the optimal length is significantly shorter or longer than this. Likewise, what is the minimal linker length for inhibitory autophosphorylation? Testing additional, intermediate linker lengths would enable you to address this – perhaps natural CaMKII isoform linkers of different lengths exist that could be tested. Also, for completeness sake, it would be ideal if you tested the reciprocal CaMKII-α/β chimera, i.e., CaMKII-α*, in which the CaMKII-α linker is swapped into CaMKII-β.The main experimental issue to be addressed is as follows. While the reviewers agree that the most interesting outcome of your analysis is the influence of the length of the catalytic domain-hub linker on the nature of the CaWe thank the reviewers for these suggestions. Using five new constructs, we have now studied the effect of kinase-hub linkers of different length on autophosphorylation outcomes in CaMKII. These results are now discussed in the manuscript  and included as new supplementary figures ). We believe that the inclusion of these results has strengthened our conclusions. A brief summary of these experimental results is presented below.1) We have now replaced the linker in CaMKII-αwith that from two other CaMKII isoforms (γwith a 110-residue linker and δwith a 31-residue linker), producing surrogate constructs, CaMKII-γ* and CaMKII-δ*. The autophosphorylation outcomes for shorter-linker CaMKII-δ* is similar to that seen for CaMKII-α, in terms of both the activating and inhibitory phosphorylation . The longer-linker CaMKII-γ* exhibits autophosphorylation status that is closer to CaMKII-β/β*, with an increased bias towards inhibitory phosphorylation . These results suggest that the longer-linker variants have an increased tendency to autophosphorylate on the inhibitory site, whereas short-linker variants are biased towards activating phosphorylation. The sequences of the linkers in the four CaMKII isoforms are very different, and so these results support the idea that the linker length is an important determinant of phosphorylation outcome.2) This conclusion was further reinforced by studying a naturally occurring splice-variant of CaMKII-β(CaMKII-β’E) that has a 55-residue linker, instead of the more common ~200 residue linker. We also designed a construct where this truncated linker from CaMKII-β’E replaced the linker in CaMKII-α(CaMKII-β’E*). For both these constructs, we see a large reduction in inhibitory phosphorylation and a small increase in activating phosphorylation when compared to normal CaMKII-β.3) As an additional control, we designed a construct of CaMKII-βin which the kinase-hub linker was replaced by that of CaMKII-α(CaMKII-α*). The autophosphorylation outcomes for CaMKII-α* is similar to that for CaMKII-α .4) We also made a construct where the entire linker in CaMKII-α is deleted (CaMKII-α-nolinker). We observe that the activating phosphorylation is reduced in this construct when compared to CaMKII-α, while the degree of pThr 305/306 remains unchanged. The deletion of the entire linker may have some steric effects that may influence the autophosphorylation outcomes, and the behavior of this construct requires more study. We have chosen not to include data for the no-linker construct in this manuscript.We agree that it would be interesting to do a more extensive study of the effects of linker length on phosphorylation outcomes, but note that this will require much more extensive experimentation than is feasible in a short time. Not only would the linker lengths have to be tested, but the sequences of the linkers would also have to be varied.Another concern is that the model still represents a rather artificial system of ectopically expressed fusion proteins. In particular, the λ phosphatase used here is known to have very broad specificity, suggesting that more relevant and selective Ser/Thr phosphatases that are known to interact and regulate CaMKII, such as PP1 and PP2A, may produce different dephosphorylation kinetics. λ phosphatase is a rather small, highly active protein of ~24 kDa, whereas the eukaryotic cell PPI and PP2A phosphatase holoenzyme complexes are significantly larger, and it is certainly possible that the kinetics of CaMKII dephosphorylation by these enzymes might be significantly slower, perhaps because of steric access issues.We thank the reviewers for raising this point. We note that we have reported dephosphorylation kinetics using purified PP1αcatalytic subunit . We do recognize that the PP1 and PP2A phosphatases in the eukaryotic cell act in concert with other regulatory subunits, and that these regulatory subunits are not included in our study. A detailed investigation of the effect of phosphatase holoenzymes is beyond the scope of the current manuscript.Reviewer #1:[…]Overall, this is a well written and detailed study. The rigorous biochemical approach of the Kuriyan lab is balanced with the power of single molecule fluorescence approaches that have been pioneered by the Grove lab. This has led to the formulation of an intricate model for the activation and inactivation of CaMKII's that may prove challenging for some readers to fully appreciate. I only have a few minor comments.1) I wonder if there is merit in incorporating Figure S3 into the body of the text. I recognize that this is already a vast study of nine full figures, but the data on dephosphorylation in a cellular context is a nice addition to the study.Thank you for this suggestion. We have now included the data on phosphorylation and dephosphorylation in the cellular context as part of a main figure .2) Figure 5 is necessary but quite complicated. I wonder if there is any way to simplify this section.Thank you for this suggestion as well. We have now revised Figure 5 to highlight only the key reaction pathways for clarity. In the figure legend, we have mentioned that this is a simplified schematic of our kinetic model and a detailed description of all the reactions is provided in the Appendix.3) Experiments with small molecule inhibitors such as staurosporine are appropriate, but I wonder if there would be benefit from including work with more established CaMKII antagonists such as KN62.We thank the reviewer for this suggestion. In the current experiment, staurosporine is used to completely abolish CaMKII kinase activity, which is achieved at the high staurosporine concentrations (100 μM) that were used. The same level of kinase inhibition can most likely be achieved at a much lower concentration with the more established CaMKII antagonists and we will integrate this idea in future investigations.Reviewer #2:[…]The reported activation and inactivation data of this key enzyme system are certainly interesting. Experimental data are sound and have been interpreted with care. My only concern is, that the model still represents a very artificial system of ectopically expressed fusion proteins. In particular, the phosphatase used is known to have very broad specificity suggesting that more relevant and selective Ser/Thr phosphatases that are known to interact and regulate CaMKII such as PP1 and PP2a, may produce different dephosphorylation kinetics. However, I think that the in vitro data are however sufficiently interesting for publication in eLife when limitations are more clearly highlighted and they may stimulate more research focussing on the role of the β isoform in more relevant cell types.We thank reviewer 2 for the positive feedback on our work. We agree that our conclusions are based on in vitroexperiments on ectopically expressed fusion proteins. Future investigations will focus on expanding the scope of our current findings to more relevant cell types and understand the physiological relevance of different CaMKII isoforms.We agree with the reviewer regarding the choice of phosphatase. We note that we have reported dephosphorylation kinetics using purified PP1 catalytic subunit . We do recognize that the PP1 and PP2A phosphatases in the eukaryotic cell act in concert with other regulatory subunits, and that these regulatory subunits are not included in our study. A detailed investigation of the effect of phosphatase holoenzymes is beyond the scope of the current manuscript.Specifically:a) The notion that α and β ratios regulate activity could be supported by (available) expression data in different cell types. Such data would at least demonstrate that the proposed regulatory mechanism is indeed observed.vice versa, resulting in functional differences between these isoforms. This difference in the expression level between two isoforms can manifest as differences in cellular targeting, stimulus frequency response, and as we now show, can switch the balance between activating and inhibitory autophosphorylation.Thank you for this suggestion. In the concluding remarks, we have now clarified that the expression levels of CaMKII-αand CaMKII-βin neurons is dynamic and is altered by synaptic activity . High neuronal activity increases the α/βratio and b) The heading "The balance between inhibitory and activating autophosphorylation is maintained upon activation of CaMKII in cells" is misleading. The experiments are carried out in lysates using ectopically expressed protein in HEK293T cells.2+. After activation, the cells are lysed, the activated holoenzymes are immobilized on the coverslip and their autophosphorylation status is directly measured without any further manipulation to the protein. To avoid confusion, we have now modified the heading slightly to say “The balance between inhibitory and activating autophosphorylation is maintained upon activation of CaMKII in HEK293T cells”. In addition, the Materials and methods section is also updated to explain this experiment more clearly.In these experiments, we are activating CaMKII variants in HEK293T cells using ionomycin and Ca2+ pulses" suggests that signalling events have been studied downstream of Ca2+ pulses – this is not the case as only dephosphorylation kinetics by l-phosphatase has been experimentally assessed.Similarly, the heading "Slower dephosphorylation at the activating site primes CaMKII for activation by incoming Ca2+ pulses have not been studied. In the revised version, we have modified the heading to say “Slower dephosphorylation at the activating site primes CaMKII for activation by subthreshold concentrations of Ca2+/CaM”.We agree with the reviewer that this heading is misleading as the signaling events downstream of the CaThe authors should also discuss that CaMKII activation in neurons is extremely transient (approximately 1 min duration). This is probably not within the time resolution of the current experimental setup.We have addressed this point in the concluding remarks (highlighted in yellow). The observation of transient activation of CaMKII in neurons relies on the use of a FRET-based sensor, Camui. Camui reports on a conformational change undergone by CaMKII during activation, and does not measure the phosphorylation states directly.Reviewer #3:[…]Although these results will be of interest to those in the CaMKII field, it is debatable whether the study, in its current form, is of high enough significance to merit publication in eLife. Given the authors' main finding that the kinase-hub linker length plays a crucial role in the activating/inhibitory phosphorylation ratio, one would think that they would have explored this feature more exhaustively, for example, by testing intermediate linker lengths . Is 30 residues (in CaMKII-α) the optimal length for Thr286 trans-autophosphorylation, or is the optimal length shorter or longer? Also, for completeness sake, the authors should test the reciprocal CaMKII-α/β chimera, i.e., CaMKII-α*, in which the CaMKII-α linker is swapped into CaMKII-β.We thank reviewer 3 for the feedback, especially highlighting that an analysis connecting the kinase-hub linker length to autophosphorylation outcomes in CaMKII is missing. In response, we have now added experimental results from five new constructs in the revised manuscript, as summarized above. We believe that the inclusion of these data in the manuscript  has strengthened the conclusions of our work. A summary of these experimental results is also presented below.1) We have now replaced the linker in CaMKII-αwith that from two other CaMKII isoforms (γwith a 110-residue linker and δwith a 31-residue linker), producing surrogate constructs, CaMKII-γ* and CaMKII-δ*. The autophosphorylation outcomes for shorter-linker CaMKII-δ* is similar to that seen for CaMKII-α, in terms of both the activating and inhibitory phosphorylation . The longer-linker CaMKII-γ* exhibits autophosphorylation status that is closer to CaMKII-β/β*, with an increased bias towards inhibitory phosphorylation . These results suggest that the longer-linker variants have an increased tendency to autophosphorylate on the inhibitory site, whereas short-linker variants are biased towards activating phosphorylation. The sequences of the linkers in the four CaMKII isoforms are very different, and so these results support the idea that the linker length is an important determinant of phosphorylation outcome.2) This conclusion was further reinforced by studying a naturally occurring splice-variant of CaMKII-β(CaMKII-β’E) that has a 55-residue linker, instead of the more common ~200 residue linker. We also designed a construct where this truncated linker from CaMKII-β’E replaced the linker in CaMKII-α(CaMKII-β’E*). For both these constructs, we see a large reduction in inhibitory phosphorylation and a small increase in activating phosphorylation when compared to normal CaMKII-β.3) As an additional control, we designed a construct of CaMKII-βin which the kinase-hub linker was replaced by that of CaMKII-α(CaMKII-α*). The autophosphorylation outcomes for CaMKII-α* is similar to that for CaMKII-α .4) We also made a construct where the entire linker in CaMKII-α is deleted (CaMKII-α-nolinker). We observe that the activating phosphorylation is reduced in this construct when compared to CaMKII-α, while the degree of pThr 305/306 remains unchanged. The deletion of the entire linker may have some steric effects that may influence the autophosphorylation outcomes, and the behavior of this construct requires more study. We have chosen not to include data for the no-linker construct in this manuscript.We agree that it would be interesting to do a more extensive study of the effects of linker length on phosphorylation outcomes, but note that this will require much more extensive experimentation than is feasible in a short time. Not only would the linker lengths have to be tested, but the sequences of the linkers would also have to be varied.


Monochamus dubius Gahan 1894. The results showed that the length of complete mitochondrial genome was 16,029 bp with 22.22% GC content, containing 39.4% A, 38.4% T, 13.4% C, 8.8% G. There were 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), two ribosomal RNA genes (rRNA) and one AT-rich region. Phylogenetic analysis showed that M. dubius was clustered with Monochamus urussovii and Monochamus alternatus alternatus, and confirmed the sister relationship among the genus Monochamus, Anoplophora, and Aristobia from Cerambycidae. The complete mitogenome of M. dubius would help understand the classification and phylogeny of Chrysomeloidea.In this study, we sequenced the complete mitochondrial genome of  M. dubius from Cerambycidae. In addition, we built the phylogenetic tree based on the maximum likelihood method to understand the evolution relationship. The results provide important information for studying the evolution of mitochondrial genomes in this group.Chrysomeloidea cover the two largest families, Cerambycidae (longhorn beetles) and Chrysomelidae (leaf beetles), each with more than 30,000 species and about a dozen subfamilies  by using sex pheromone traps. The specimens were deposited at −80 °C in the Key Laboratory of Integrated Pest Management in Ecological Forests, Fujian Agriculture and Forestry University  under the voucher number TN-202009. The total DNA was extracted from the legs using Insect DNA Kit  and purified using QIAquick Gel Extraction Kit . The mitochondrial genome was sequenced by the Illumina Hiseq 2500  at the Novogene . A total of 66,458,876 clean reads were obtained by quality control and filtration from the 68,684,628 raw reads. After the de novo assembly of MitoZ and metaSpades software  as an out-group, a maximum likelihood analysis of 11 Cerambycidae species and 6 Chrysomelidae species was performed with the software MEGA 7.0, following by calculating the bootstrap values with 1000 replications (Kumar et al. M. dubius constituted a monophyletic group with other 10 Cerambycidae. The monophyletic Cerambycidae that contains Lamiinae, Aseminae, Spondylinae and Lepturinae was assigned to the sister group to the clade of Chrysomelidae that consists of Chrysomelinae and Galerucinae in this study. Additionally, M. dubius was clustered together with M. urussovii and M. alternatus alternatus (M. dubius will provide useful genetic information for the genetic evolution of M. dubius, as well as in other insects of Chrysomeloidea.To investigate its taxonomic status of ternatus . The com