A calcium/calmodulin-binding serine/threonine protein kinase homologous to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells.

cDNA fragments corresponding to an apple (Malus domestica [L.] Borkh) calmodulin-binding polypeptide have been isolated and characterized. The protein encoded by this messenger contains a serine/threonine protein kinase catalytic domain followed by a calcium/calmodulin-binding regulatory domain, both exhibiting significant sequence similarities to the corresponding regions of the mammalian calcium/calmodulin-dependent protein kinase II subunits. These results confirm a potential regulatory role for calmodulin in phosphorylation-mediated signal transduction events.     

Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.     

Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.     

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Br?nsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

丝氨酸/苏氨酸蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用

Tau蛋白过度磷酸化在阿尔采末病（Alzheimer’s disease,AD）发病过程中发挥重要作用,抑制蛋白磷酸酯酶活性,可诱导tau的过度磷酸化和聚积。本文拟就近年来蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用作一综述。     

Role of serine/threonine protein phosphatase in Alzheimer's disease

Accumulating evidence indicates that serine/threonine (Ser/Thr) protein phosphatases (PPs), such as PP1, PP2A and PP2B, participate in the neurodegenerative progress in Alzheimer's disease (AD). The general characteristics and pathologic changes of PP1, PP2A and PP2B in AD, and their relations with microtubule-associated proteins, focusing mainly on tau protein, neurofilament (NF), amyloid precursor protein (APP) processing and synaptic plasticity are discussed. Deriving novel insight into the particular topic will attract greater attention to more active investigation and effective therapeutic intervention in the future.     

Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.     

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca²⁺/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca²⁺/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein–protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database (https://www.wikipathways.org/index.php/Pathway:WP4874). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00592-1) contains supplementary material, which is available to authorized users.