Search

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into different classes based on their processing enzymes. The three-domain class IV lanthipeptide synthetases (LanL enzymes) consist of N-terminal lyase, central kinase, and C-terminal cyclase domains. While the catalytic residues of the kinase domains (mediating ATP-dependent Ser/Thr phosphorylations) and the lyase domains (carrying out subsequent phosphoserine/phosphothreonine (pSer/pThr) eliminations to yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues) have been characterized previously, such studies are missing for LanL cyclase domains. To close this gap of knowledge, this study reports on the identification and validation of the catalytic residues in the cyclase domain of the class IV lanthipeptide synthetase SgbL, which facilitate the nucleophilic attacks by Cys thiols on Dha/Dhb residues for the formation of β-thioether crosslinks.

Dual‐specificity phosphatases (DUSPs) are defined by their capability to dephosphorylate both phosphoserine/phosphothreonine (pSer/pThr) and phosphotyrosine (pTyr). DUSP5, a member of DUSPs superfamily, is located in the nucleus and plays crucially regulatory roles in the signaling pathway transduction. In our present study, we discover that DUSP5 significantly promotes osteogenic differentiation of mesenchymal stromal cells (MSCs) by activating SMAD1 signaling pathway. Mechanistically, DUSP5 physically interacts with the phosphatase domain of small C‐terminal phosphatase 1/2 (SCP1/2, SMAD1 phosphatases) by the linker region. In addition, we further confirm that DUSP5 activates SMAD1 signaling through a SCP1/2‐dependent manner. Specifically, DUSP5 attenuates the SCP1/2‐SMAD1 interaction by competitively binding to SCP1/2, which is responsible for the SMAD1 dephosphorylation, and thus results in the activation of SMAD1 signaling. Importantly, DUSP5 expression in mouse bone marrow MSCs is significantly reduced in ovariectomized (OVX) mice in which osteogenesis is highly passive, and overexpression of Dusp5 via tail vein injection reverses the bone loss of OVX mice efficiently. Collectively, this work demonstrates that the linker region of DUSP5 maybe a novel chemically modifiable target for controlling MSCs fate choices and for osteoporosis treatment., DUSP5 promotes the osteogenic differentiation of mesenchymal stromal cells (MSCs) by repressing SMAD1 signaling pathway in a SCP1/2‐dependent manner. The linker region of DUSP5 occupies the phosphatase domain of SCP1/2 and thereby releases the inhibitory effect of SCP1/2 on SMAD1 signaling. Additionally, Dusp5 overexpression could effectively ameliorate osteopenia of mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease, but its underlying mechanism is still unclear and the identities of drugs for AD also lack. Tau acetylation has become potentially important post-translational modification of tau. Levels of tau acetylation are significantly enhanced in AD patients and transgenic mouse models of AD, but the underlying mechanism and roles of tau hyperacetylation in AD onset maintain elusive. In the current study, we found that tau acetylation is obviously enhanced and the activities of AMP-activated protein kinase (AMPK) and sirtuin1 (Sirt1) are significantly decreased in APP/PS1 and streptozotocin (STZ) mice and high glucose (HG)-treated cells. Moreover, we demonstrated that activation of AMPK reduces the level of tau acetylation and ameliorates memory impairment, and its mechanism is associated with activation of Sirt1. Taken together, AMPK might be a crucial upstream molecular to regulate acetylation of tau and become a new target for AD therapy in the future.

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function. Electronic supplementary material The online version of this article (10.1007/s12035-020-02038-6) contains supplementary material, which is available to authorized users.

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences., The substrate specificity of phosphoprotein phosphatases PP1 and PP2A depends on their catalytic and regulatory subunits. Using proteomics approaches, the authors here provide insights into the sequence specificity of the catalytic subunits and their distinct contributions to PP1 and PP2A selectivity.

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1β and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1β induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1β-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.

Primary cilia (PC) are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling final step of cilia initiation. The function of TTBK2 in ciliogenesisis is critically dependent on its kinase activity, however, precise mechanism of TTBK2 action is so far incompletely understood, due to very limited information about its relevant substrates. In this study we identify CEP83, CEP89, CCDC92, Rabin8 and DVL3 as substrates of TTBK2 kinase activity. Further, we characterise a set of phosphosites of the newly identified substrates and CEP164, induced by TTBK2in vitroandin vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we address functional relevance of selected phosphorylations of CEP164 and provide evidence that the examined TTBK2-induced phosphorylations of CEP164 are relevant for the process of cilia formation. In summary, our work provides important insight into substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

Background Eukaryotic elongation factor-2 kinase (eEF2K) is a Ca 2+ /calmodulin (CaM)-dependent protein kinase that inhibits protein synthesis. However, the role of eEF2K in cancer development was reported paradoxically and remains to be elucidated. Methods Herein, A549 cells with eEF2K depletion or overexpression by stably transfected lentivirus plasmids were used in vitro and in vivo study. MTT and colony assays were used to detect cell proliferation and growth. Extracellular glucose and lactate concentration were measured using test kit. Immunoblot and co-immunoprecipitation assays were used to examine the molecular biology changes and molecular interaction in these cells. LC-MS/MS analysis and [γ- 32 P] ATP kinase assay were used to identify combining protein and phosphorylation site. Nude mice was utilized to study the correlation of eEF2K and tumor growth in vivo. Results We demonstrated that eEF2K inhibited lung cancer cells proliferation and affected the inhibitory effects of EGFR inhibitor gefitinib. Mechanistically, we showed that eEF2K formed a complex with PKM2 and STAT3, thereby phosphorylated PKM2 at T129, leading to reduced dimerization of PKM2. Subsequently, PKM2 impeded STAT3 phosphorylation and STAT3-dependent c-Myc expression. eEF2K depletion promoted the nuclear translocation of PKM2 and increased aerobic glycolysis reflected by increased lactate secretion and glucose. Conclusions Our findings define a novel mechanism underlying the regulation of cancer cell proliferation by eEF2K independent of its role in protein synthesis, disclosing the diverse roles of eEF2K in cell biology, which lays foundation for the development of new anticancer therapeutic strategies.

The large families of amastins from Leishmania donovani, L. infantum, L. major, L. braziliensis and Trypanosoma cruzi are strongly associated with the evolution of intracellular parasitism of rich cells in human MHC.1 molecules such as the macrophages, dendritic cells, and Langerhans cells by these parasites, recognize the MHC-1 molecules as host receptor. The internalization and transport of the paraste in the cytoplas of infected cell is facilitated by the MHC-1 recycle and endosome formation drag and transport the parasite in the cytoplasm of infected cell. The microbody amastins participate as coreceptor potency the infection, the tropism of L. major and L. braziliensis by the cells from the skin is facilitated by two molecular interactions, the first molecular interaction is faclitated by the amastins interact the human MHC-1 molecules, and the second molecular interaction is facilitated by the numerous microbody amastins; which also participate in the biogenesis of the small prasitophorous vcuole from L. major, and large parasitophorous vacuole from L. braziliensis.All amastins from these parasites developed deactivation domains, in different grade L. donovani develop an amastin surface coat specialized in deactivation of infected macrophages heavily glycosylated developed 38 amastins with 38 glycosylation Asp. N-Glycosylation sites and 45 N-glucosamina glycosylation sites, whereas L. infantum, L. major and L. braziliensis developed one half of glycosylated amastins in asparagine N-glycosylation sites, and T. cruzi did not developed none glycosylated amastin.The amastins surface coat from L. donovani is rich in phosphorylation sites, developed 45 amastins with 45 casein kinase II phosphorylations sites, and 48 amastins with 48 protein kinase phosphorylation sites. L. infantum, L. braziliensis, and T. cruzi developed 32, 42, and 8 amastins, with 94, 114, 21 casein kinase II phosphorylation sites; in similar way developed 35,38, 11 amastins with 89,78, and 22 protein kinase phosphorylation sites. The family of amastins from L. donovani develop 137 phosphoserines. and 128 phosphothreonine, L. major developed 14 phosphoserine and 4 phosphothreonine; L. infantum 1 phophoserine and 7 phosphothreonine; L. braziliensis did not developed phosphoserine and phosphothreonine and T. cruzi 4 phosphoserine and 4 phosphothreonine. The results show that amastin surface coat is equiped with numerous phosphorylations sites atractive for phosphohrylases from the infected host contribute with the dephosphorylation and deactivation of infectetd host cells.The amastins from L. major develop a membrane amastin with laminin G domain, which can interact with the collagen and heparin sulfate proteoglycan sites from the extracellular matrix of the skin tissue. Furthermore develop 14 amastins with tyrosine sulfation site, evade the activation of receptor of chemokines and the activation of the immune response by chemokines.There is an alternative mechanism of polarization of the immune response from protective TH1 to non protective TH2.The parasite nutrition is mediated by amastins that dissimilate the MHC-1 molecules and other subsets of proteins, the dissimilation products can be translocated through of the parasite cell membrane and employed as nutrient source.

Targeting protein – protein interactions (PPIs) has emerged as an important area of discovery for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the polo-like kinase 1 (Plk1) polo-box domain (PBD), a phosphorylated protein residue can provide high-affinity recognition and binding to target protein hot spots. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden “cryptic” pockets that may be revealed on ligand binding. In our current paper, we describe the design and synthesis of macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a phosphothreonine (pT) residue for optimal interaction in the signature phospho-binding pocket. Macrocycles prepared with this new amino acid analog introduce additional hydrogen-bonding interactions not found in the open-chain linear parent peptide. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets., A novel macrocyclic peptide strategy utilizing a glutamic acid analog that can work as an alkylated histidine replacement.

Phosphorylation, which is mediated by protein kinases and opposed by protein phosphatases, is an important post-translational modification that regulates many cellular processes, including cellular metabolism, cell migration, and cell division. Due to its essential role in cellular physiology, a great deal of attention has been devoted to identifying sites of phosphorylation on cellular proteins and understanding how modification of these sites affects their cellular functions. This has led to the development of several computational methods designed to predict sites of phosphorylation based on a protein’s primary amino acid sequence. In contrast, much less attention has been paid to dephosphorylation and its role in regulating the phosphorylation status of proteins inside cells. Indeed, to date, dephosphorylation site prediction tools have been restricted to a few tyrosine phosphatases. To fill this knowledge gap, we have employed a transfer learning strategy to develop a deep learning-based model to predict sites that are likely to be dephosphorylated. Based on independent test results, our model, which we termed DTL-DephosSite, achieved efficiency scores for phosphoserine/phosphothreonine residues of 84%, 84% and 0.68 with respect to sensitivity (SN), specificity (SP) and Matthew’s correlation coefficient (MCC). Similarly, DTL-DephosSite exhibited efficiency scores of 75%, 88% and 0.64 for phosphotyrosine residues with respect to SN, SP, and MCC.

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits are known to engage PP1c’s surface, locally affecting flexible phosphopeptides access to the active site. However, catalytic efficiency of holophosphatases towards their natively-folded phosphoprotein substrates is largely unexplained. Here we present a Cryo-EM structure of the tripartite PP1c/PPP1R15A/G-actin holophosphatase that terminates signalling in the Integrated Stress Response (ISR) in pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin’s role in eIF2α dephosphorylation is supported crystallographically by the structure of the binary PPP1R15A-G-actin complex, and by biochemical and genetic confirmation of the essential role of PPP1R15A-G-actin contacts to eIF2αP dephosphorylation. In the pre-dephosphorylation CryoEM complex, G-actin aligns the catalytic and regulatory subunits, creating a composite surface that engages eIF2α’s N-terminal domain to position the distant phosphoserine-51 at the active site. eIF2α residues specifying affinity for the holophosphatase are confirmed here to make critical contacts with the eIF2α kinase PERK. Thus, a convergent process of higher-order substrate recognition specifies functionally-antagonistic phosphorylation and dephosphorylation in the ISR.

Highlights • Phosphorylome of Trichoderma reesei reveals phosphosites in some glycosyl hydrolases. • Phosphoserine and phosphothreonine is the major phosphosites identified. • Protein Kinase C is the most frequently predicted kinase in phosphorylome. • The cellulase Cel7A activity is affected by dephosphorylation., Trichoderma reesei is one of the major producers of holocellulases. It is known that in T. reesei, protein production patterns can change in a carbon source-dependent manner. Here, we performed a phosphorylome analysis of T. reesei grown in the presence of sugarcane bagasse and glucose as carbon source. In presence of sugarcane bagasse, a total of 114 phosphorylated proteins were identified. Phosphoserine and phosphothreonine corresponded to 89.6% of the phosphosites and 10.4% were related to phosphotyrosine. Among the identified proteins, 65% were singly phosphorylated, 19% were doubly phosphorylated, 12% were triply phosphorylated, and 4% displayed even higher phosphorylation. Seventy-five kinases were predicted to phosphorylate the sites identified in this work, and the most frequently predicted serine/threonine kinase was PKC1. Among phosphorylated proteins, four glycosyl hydrolases were predicted to be secreted. Interestingly, Cel7A activity, the most secreted protein, was reduced to approximately 60% after in vitro dephosphorylation, suggesting that phosphorylation might alter Cel7A structure, substrate affinity, and targeting of the substrate to its carbohydrate-binding domain. These results suggest a novel post-translational regulation of Cel7A.

Thylakoid membranes in land plant chloroplasts are organized into appressed and nonappressed membranes, which contribute to the control of energy distribution between the two photosystems (PSI and PSII) from the associated light-harvesting complexes (LHCs). Under fluctuating light conditions, fast reversible phosphorylation of the N-terminal thylakoid protein domains and changes in electrostatic forces induce modifications in thylakoid organization. To gain insight into the role and dynamics of thylakoid protein phosphorylation, we used targeted proteomics to quantify amounts of the structural proteins CURVATURE THYLAKOID1 (CURT1), including the levels of CURT1B N terminus phosphorylation and acetylation, after short-term fluctuating light treatments of Arabidopsis (Arabidopsis thaliana). The CURT1B protein was localized to a specific curvature domain separated from the margin domain, and specifically depleted of chlorophyll-binding protein complexes. The acetylation and phosphorylation of the CURT1B N terminus were mutually exclusive. The level of CURT1B phosphorylation, but not of acetylation, increased upon light shifts that also led to an increase in PSII core protein phosphorylation. These dynamics were largely absent in the knockout mutant of PSII core protein kinase SER/THR PROTEIN KINASE8 (STN8). Moreover, in mutants impaired in interaction between phosphorylated LHCII and PSI, the phosphorylation dynamics of CURT1B and the amount of the other CURT1 proteins were misregulated, indicating a functional interaction between CURT1B and PSI-LHCII complexes in grana margins. The complex relationships between phosphorylation of PSII, LHCII, and CURT1B support the dynamics of thylakoid protein complexes that are crucial in the optimization of photosynthesis under fluctuating light intensities.

Introduction The endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. Methods Rat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1 mg/L, 1 mg/L, and 10 mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. Results LPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. Conclusion Phosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.

Granulocyte colony stimulating factor receptor (G-CSFR) plays an important role in the production of neutrophil granulocytes. Mutated G-CSFRs have been directly associated with two distinct malignant phenotypes in patients, e.g. acute myeloid leukemia (AML) and chronic neutrophilic leukemia (CNL). However, the signaling mechanism of the mutated G-CSFRs is not well understood. Here, we present a comprehensive SILAC-based quantitative phosphoserine and phosphothreonine dataset of the normal and mutated G-CSFRs signaling using the BaF3 cell-line-based in vitro model system. High pH reversed phase concatenation and Titanium Dioxide Spin Tip column were utilized to increase the dynamic range and detection of the phosphoproteome of G-CSFRs. The dataset was further analyzed using several computational tools to validate the quality of the dataset. Overall, this dataset is the first global phosphoproteomics analysis of both normal and disease-associated-mutant G-CSFRs. We anticipate that this dataset will have a strong potential to decipher the phospho-signaling differences between the normal and malignant G-CSFR biology with therapeutic implications. The phosphoproteomic dataset is available via the PRIDE partner repository., Design Type(s)protein physical property analysis objective • replicate design • factorial designMeasurement Type(s)protein expression profilingTechnology Type(s)liquid chromatography-tandem mass spectrometryFactor Type(s)biological replicate • experimental conditionSample Characteristic(s)Mus musculus • BA/F3 cell Machine-accessible metadata file describing the reported data (ISA-Tab format)

The dynamics of caseinophosphopeptides (CPP) in simulated gastrointestinal digestion was studied. CPP in the undigested milk and in vitro simulated gastrointestinal hydrolysates were enriched using immobilized titanium(IV) ion affinity chromatography (Ti4+-IMAC) beads and measured using liquid chromatography-tandem mass spectrometry. There were 62, 80 and 38 CPP identified from undigested milk, simulated gastric and pancreatic hydrolysates, respectively. In total, 142 unique CPP and 27 casein phosphorylation sites were identified. Only 5 of 62 endogenous CPP in undigested milk survived in the simulated gastrointestinal hydrolysates and these 5 CPP were semi-quantified. Most of the CPP in undigested milk came from β-casein. Further, 13 CPP with phosphothreonine were found in simulated gastrointestinal hydrolysates. Seven CPP with the conserved sequence SpSpSpEE were identified from simulated gastrointestinal hydrolysates. Only 2 of the 6 CPP with the conserved sequence SpSpSpEE in undigested milk survived in the simulated gastric digestion, and all of them were digested by pancreatin during simulated pancreatic digestion. The results showed that most of the CPP were released from large endogenous CPP or caseins in simulated gastrointestinal hydrolysates. However, most of them were degraded after simulated pancreatic digestion while the content of some persistent CPP increased at the same time.

Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a distinct mechanism from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex may represent an attractive therapeutic target in late-stage malignancies.SummaryGemperle et al. present the first report of an interaction between p130Cas with the serine/threonine kinase PKN3, implicated in prostate and breast cancer growth. They show that p130Cas colocalizes with PKN3 in cell structures that have a pro-invasive function and enhance our understanding of PKN3-mediated signaling and tumor growth.

We herein report two examples of one-pot, simultaneous reactions, mediated by multiple, orthogonal catalysts with the same catalytic motif. First, BINOL-derived chiral phosphoric acids (CPA) and phosphothreonine (pThr)-embedded peptides were found to be matched for two different steps in double reductions of bisquinolines. Next, two π-methylhistidine (Pmh)-containing peptides catalyzed enantio- and chemoselective acylations and phosphorylations of multiple substrates in one pot. The selectivity exhibited by common reactive moieties is adjusted solely by the appended chiral scaffold through outer-sphere interactions.

We report a facile synthetic strategy toward CH2-substituted phosphothreonine mimetics. Herein, inexpensive valine with a directing group was converted into homothreonine via palladium-catalyzed γ-methyl C(sp3)–H bond activation, followed by construction of a phosphorus–carbon bond via the well-developed Appel reaction and Michaelis–Becker reaction with a total yield of 30%. Furthermore, the derived mimetic was applied for solid-phase synthesis of two phosphopeptide inhibitors. This efficient synthesis provides a chance to prepare not only phosphopeptides but also phosphoproteins resistant to phosphatases.

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

Ca2+/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment also induces the phosphorylation in the activation loop of OsMKK1, and the two phosphorylations in the N-terminus and in the activation loop are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in the activation of MAPK cascade and ABA signaling.One-sentence summaryOsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in the activation of MAPK cascade and ABA signaling.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Mingyi Jiang (myjiang@njau.edu.cn)

Protein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH ≤ 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells.

The strong association between diabetes mellitus type 2 and cancer is observed. The incidence of both diseases is increasing globally due to the interaction between them. Recent studies suggest that there is also an association between cancer incidence and anti-diabetic medications. An inhibitor of dipeptidyl-peptidase 4 (DPP-4), sitagliptin, is used in diabetes treatment. We examined the influence of sitagliptin alone or in combination with a cytostatic drug (paclitaxel) on the development of epithelial ovarian cancer cells and the process of metastasis. We examined migration, invasiveness, apoptosis, and metalloproteinases (MMPs) and their inhibitors&rsquo, (TIMPs) production in two human ovarian cancer cell lines. Sitagliptin induced apoptosis by caspase 3/7 activation in paclitaxel-treated SKOV-3 and OVCAR-3 cells. Sitagliptin maintained paclitaxel influence on ERK and Akt signaling pathways. Sitagliptin additionally reduced migration and invasiveness of SKOV-3 cells. There were distinct differences of metalloproteinases production in sitagliptin-stimulated ovarian cancer cells in both cell lines, despite their identical histological classification. Only the SKOV-3 cell line expressed MMPs and TIMPs. SKOV-3 cells co-treated with sitagliptin and paclitaxel decreased concentrations of MMP-1, MMP-2, MMP-7, MMP-10, TIMP-1, TIMP-2. The obtained data showed that sitagliptin used with paclitaxel may be considered as a possibility of pharmacological modulation of intracellular transmission pathways to improve the response to chemotherapy.

Summary: The glyoxalase system is a highly conserved and ubiquitously expressed enzyme system, which is responsible for the detoxification of methylglyoxal (MG), a spontaneous by-product of energy metabolism. This study is able to show that a phosphorylation of threonine-107 (T107) in the (rate-limiting) Glyoxalase 1 (Glo1) protein, mediated by Ca2+/calmodulin-dependent kinase II delta (CamKIIδ), is associated with elevated catalytic efficiency of Glo1 (lower KM; higher Vmax). Additionally, we observe proteasomal degradation of non-phosphorylated Glo1 via ubiquitination does occur more rapidly as compared with native Glo1. The absence of CamKIIδ is associated with poor detoxification capacity and decreased protein content of Glo1 in a murine CamKIIδ knockout model. Therefore, phosphorylation of T107 in the Glo1 protein by CamKIIδ is a quick and precise mechanism regulating Glo1 activity, which is experimentally linked to an altered Glo1 status in cancer, diabetes, and during aging.

RATIONALE: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. OBJECTIVE: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. METHODS AND RESULTS: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Single Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p

Biomolecules undergo liquid-liquid phase separation (LLPS) resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed charge atomistic forcefields, we parameterize the size and atomistic hydropathy of the coarse-grained modified amino acid beads, and hence the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein’s single chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of FUS and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.Statement of SignificancePost-translational modifications are important regulators of liquid-liquid phase separation (LLPS) which drives the formation of biomolecular condensates. Theoretical methods can be used to characterize the biophysical properties of intrinsically disordered proteins (IDPs). Our recent framework for molecular simulations using a Cα-centered coarse-grained model can predict the effect of various perturbations such as mutations (Dignon et al. PloS Comput. Biol, 2018) and temperature (Dignon et al, ACS Cent. Sci., 2019) on LLPS. Here, we expand this framework to incorporate modified residues like phosphothreonine, phosphoserine and acetylysine. This model will prove useful for simulating the phase separation of post-translationally modified IDPs and predicting how position-specific modifications can control phase behavior across the large family of proteins known to be phosphorylated and acetylated.

Cross‐linking of the B‐cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post‐translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death‐associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti‐IgM‐dependent manner. DAPK inhibition mimics ibrutinib‐induced repression of both IEG mRNA and histone H3 phosphorylation and has anti‐proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti‐tumour effect than ibrutinib, by repressing both anti‐IgM‐ and CD40L‐dependent activation., Transcription involves alteration of histones, the proteins surrounding DNA. Here, we show that phosphorylation of histone H3 mediated by the protein DAPK3 does not impact on transcription itself, but is required for co‐transcriptional mRNA processing of key pro‐proliferative genes. Inactivation of DAPK3 not only prevents mRNA processing of these genes but correlates with accumulation of their unprocessed pre‐mRNA transcripts.

Mutations in genes that encode components of tuberous sclerosis complex 2 (TSC2) are associated with tuberous sclerosis complex disease. TSC2 interacts with tuberous sclerosis complex 1 to form a complex that negatively regulates cell growth and proliferation via the inactivation of mechanistic target of rapamycin complex 1. The activity of TSC2 is mainly regulated via posttranslational modifications such as phosphorylation. However, the control of TSC2 activity is not entirely achieved by phosphorylation. In this study, we show that TSC2 is methylated at R1457 and R1459 by protein arginine methyltransferase 1 (PRMT1). Methylation of these two residues can affect the phosphorylation status through protein kinase B (Akt) of TSC2 at T1462 and is essential for TSC2 stability. Taken together, these findings indicate that novel posttranslational modifications are important for the regulation of TSC2 stability through PRMT1-mediated methylation.

An et al. identify a novel MST4-mediated YAP inactivation signaling pathway and demonstrate that the MST4–YAP axis is required to suppress gastric tumorigenesis in vivo., Hyperactivation of YAP has been commonly associated with tumorigenesis, and emerging evidence hints at multilayered Hippo-independent regulations of YAP. In this study, we identified a new MST4–YAP axis, which acts as a noncanonical Hippo signaling pathway that limits stress-induced YAP activation. MST4 kinase directly phosphorylated YAP at Thr83 to block its binding with importin α, therefore leading to YAP cytoplasmic retention and inactivation. Due to a consequential interplay between MST4-mediated YAP phospho-Thr83 signaling and the classical YAP phospho-Ser127 signaling, the phosphorylation level of YAP at Thr83 was correlated to that at Ser127. Mutation of T83E mimicking MST4-mediated alternative signaling restrained the activity of both wild-type YAP and its S127A mutant mimicking loss of classical Hippo signal. Depletion of MST4 in mice promoted gastric tumorigenesis with diminished Thr83 phosphorylation and hyperactivation of YAP. Moreover, loss of MST4–YAP signaling was associated with poor prognosis of human gastric cancer. Collectively, our study uncovered a noncanonical MST4–YAP signaling axis essential for suppressing gastric tumorigenesis., Graphical Abstract

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility., K + channels function in macromolecular complexes with accessory subunits to regulate neuronal function. Here, the authors describe Pin1-mediated regulation of the Kv4.2 complex, which impacts reversal learning in mice, providing potential treatment for disorders characterized by cognitive inflexibility.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restrictedϕ(ϕ∼ –60°) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n→π* interaction between consecutive carbonyls, and an n→σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C–Hβ that restricts theχ2side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline’s backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine areϕ,ψ= polyproline helix or α-helix (ϕ∼ –60°);χ1=g−;χ2= eclipsed C–H/O–P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation (p-BimEL-S65, p-BimEL-T112) in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors., The BRCA2 tumour suppressor protein is known to play an important role in homologous recombination. Here the authors reveal how the phosphorylation of BRCA2 by Polo-like kinase 1 (PLK1) contributes to the regulation of mitosis.

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.

A novel and facile synthetic strategy for α,α-difluorinated phosphonate mimetics of phosphoserine/phosphothreonine utilizing rhodium-catalyzed asymmetric hydrogenation was developed. The dehydrogenated substrate β-difluorophosphonomethyl α-(acylamino)acrylates were first prepared from protected serine/threonine followed by asymmetric hydrogenation using the rhodium-DuPhos catalytic system to generate the chiral center(s). These important phosphonate building blocks were successfully incorporated into phosphatase-resistant peptides, which displayed similar inhibition to the 14-3-3 ζ protein as the parent pSer/pThr peptides.

One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the nondried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in the presence of hydroxylamine promotes β-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6-fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15000 phosphopeptides from 3 mg TMT 6-plex labeled-peptides.

Summary Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

Although protein crosslinking is often linked with aging as well as some age-related diseases, very few molecular details are available on the nature of the amino acids involved, or mechanisms that are responsible for crosslinking. Recent research has shown that several amino acids are able to generate reactive intermediates that ultimately lead to covalent crosslinking through multiple non-enzymatic mechanisms. This information has been derived from proteomic investigations on aged human lenses and the mechanisms of crosslinking, in each case, have been elucidated using model peptides. Residues involved in spontaneous protein-protein crosslinking include aspartic acid, asparagine, cysteine, lysine, phosphoserine, phosphothreonine, glutamic acid and glutamine. It has become clear, therefore, that several amino acids can act as potential sites for crosslinking in the long-lived proteins that are present in aged individuals. Moreover, the lens has been an invaluable model tissue and source of crosslinked proteins from which to determine crosslinking mechanisms that may lead to crosslinking in other human tissues.

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA–FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors.

BACKGROUND Gastric cancer (GC) is the second leading cause of cancer-related death worldwide, but little progress has been achieved in the treatment of advanced or metastatic GC. GC is highly heterogeneous and more studies are needed to elucidate the metastatic mechanisms. Epithelial cell transforming 2 (ECT2) has been reported to be up-regulated in GC tissues, but its signaling mechanisms remain unclear. MATERIAL AND METHODS In this study, we used Western blot analysis to compare the expression level of ECT2 in 2 GC cell lines: MKN1 and MKN45. Mutagenesis and transfections were conducted to investigate the oncogenic mechanisms of ECT2 in GC cells. RESULTS ECT2 was expressed at higher levels in MKN1 than in MKN45. Immunoblotting results showed that MKN1 expression was suppressed by p53-WT but was enhanced by p53-mutant. In addition, in vitro experiments showed that ECT2 positively regulated the proliferation and invasion of GC cells. To better explore the mechanisms of ECT2 in promoting GC progression, we introduced site-directed mutants of ECT2, and found that the phosphor-mimic mutant T359D enhanced its oncogenic activity. In contrast, activation of RhoA was inhibited in cells transfected with ECT2 phosphor-deficient mutant T359A. We found that the epithelial cell biomarker E-cadherin was down-regulated by ECT2-T359D, highlighting the role of phosphorylation in regulating epithelial-mesenchymal transition. CONCLUSIONS Our results identified p53 as a novel up-stream signaling molecule of ECT2 in GC cells, and the post-translational modifications of ECT2 play important roles in regulating cancer development and progression.

Our previous study demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF) induced tyrosine phosphorylation/activation of the EGF receptor (EGFR). This paper was designed to investigate the mechanism of the inhibition of PKG II on EGFR activation. Gastric cancer cells HGC-27 and AGS were infected with an adenoviral vector encoding the cDNA of PKG II (Ad-PKG II) to overexpress PKG II and treated with 8-(4-chlorophenylthio) guanosine-3′,5′-cyclic monophosphate (8-pCPT-cGMP) to activate the kinase. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay were performed to detect the interaction between PKG II and EGFR. Western blotting, mass spectrometry (MS) and site mutagenesis were performed to detect the PKG II-specific phosphorylation site on EGFR. The results showed that in living COS-7 cells, which were infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was an interaction between PKG II and EGFR. The results also showed that PKG II caused threonine 669 (T669) phosphorylation of EGFR in HGC-27 and AGS cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then inhibited the activation of EGFR. When T669 of EGFR was mutated to alanine, the inhibitory effect of PKG II on the activation of EGFR was eradicated. These findings suggested a PKG II-specific phosphorylation site on EGFR, and might be beneficial to illuminate the anti-tumor role of PKG II.

Plant stress responses involve dynamic growth regulation. Growth is restricted in harsh environmental conditions and is rapidly restored when conditions improve. Here, we identified BIN2, a glycogen synthase kinase 3 (GSK3)-like kinase, as a molecular switch in the transition to robust growth after salt stress in Arabidopsis thaliana. In the rapid recovery phase after salt stress, the calcium sensors SOS3 and SCaBP8 perceive a calcium signal and promote BIN2 localization to the plasma membrane to repress the salt stress response, and BIN2 inhibits SOS2 activity and enhances growth by releasing BZR1/BES1 transcriptional activity. The expression of stress- and brassinosteroid-responsive genes is coordinately regulated during this process. bin2-3bil1 and bin2-3bil2 mutants defective in BIN2 and its homologs BIL1 and BIL2, respectively, are hyposensitive to salt stress. Our study suggests that salt signaling modulates the subcellular localization and interactions of BIN2. By phosphorylating different substrates, BIN2 regulates the salt stress response and growth recovery.

The androgen receptor (AR) is often activated in prostate cancer patients undergoing androgen-ablative therapy because of the activation of cellular pathways that stimulate the AR despite low androgen levels. In many of these tumors, the cAMP-dependent protein kinase A (PKA) pathway is activated. Previous studies have shown that PKA can synergize with low levels of androgen to enhance androgen signaling and consequent cell proliferation, leading to castration-resistant prostate cancer. However, the mechanism by which PKA causes AR stimulation in the presence of low/no androgen is not established yet. Here, using immunofluorescence immunoblotting assays, co-immunoprecipitation, siRNA-mediated gene silencing, and reporter gene assays, we demonstrate that PKA activation is necessary for the phosphorylation of heat shock protein (HSP90) that binds to unliganded AR in the cytoplasm, restricting its entry into the nucleus. We also found that PKA-mediated phosphorylation of the Thr