Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH(2)-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH(2)-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.
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PMID:Mutagenesis analysis of human SM22: characterization of actin binding. 1105 53

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.
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PMID:Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation. 1110 20

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.
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PMID:Phosphorylation of protein phosphatase inhibitor-1 by Cdk5. 1127 34

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Here we identify Snapin, a protein of relative molecular mass 15,000 that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolysable cAMP analogue induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. Our results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway.
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PMID:Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex. 1128 5

Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-independent but stimulated by cAMP. Recombinant protein kinase A (PKA) could phosphorylate phogrin directly. High performance liquid chromatography analysis of tryptic phosphopeptides, combined with site-directed mutagenesis of candidate sites, revealed the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680 and Thr-699 had been replaced either by alanines or by aspartic acid residues, were targeted to secretory granules in transfected AtT20 neuroendocrine cells. Stimulation of these cells with a range of secretagogues, including K(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.
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PMID:Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase. 1135 72

Xenopus early response 1 (XER1) is a fibroblast growth factor-inducible transcription factor whose developmentally regulated nuclear localization is thought to be important in the control of cell differentation during embryonic development [Luchman et al., Mech. Dev. 80 (1999) 111-114]. Analysis of the XER1 amino acid sequence revealed four regions which contain potential nuclear localization sequences (NLSs). Using mutant XER1 proteins and portions of XER1 fused to green fluorescent protein (GFP) transfected into NIH 3T3 cells, we have determined that only one of these, NLS4, located near the carboxy-terminus of XER1, is necessary and sufficient for targeting exclusively to the nucleus. Of the other three predicted NLS sequences, only NLS1, consisting of the sequence (138)RPRRCK(143) was shown to function as a cryptic, weak NLS. NLS4 contains a core region consisting of the sequence (463)RPIKRQRMD(471) which is similar to the core NLS directing the human c-MYC protein to the nucleus. The core sequence is flanked by a predicted cdc2/protein kinase A phosphorylation motif, however mutation of the serine(472) to alanine or aspartic acid had no detectable effect on accumulation of GFP-XER1 fusion proteins in the nucleus, demonstrating that this putative phosphorylation site plays no role in regulating nuclear transport.
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PMID:Nuclear localization signals in the Xenopus FGF embryonic early response 1 protein. 1147 45

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.
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PMID:Caspase-mediated cleavage of the Ca2+/calmodulin-dependent protein kinase-like kinase facilitates neuronal apoptosis. 1147 89

Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.
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PMID:Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. 1156 17

Death-associated protein kinase is a calcium/calmodulin serine/threonine kinase, which positively mediates programmed cell death in a variety of systems. Here we addressed its mode of regulation and identified a mechanism that restrains its apoptotic function in growing cells and enables its activation during cell death. It involves autophosphorylation of Ser(308) within the calmodulin (CaM)-regulatory domain, which occurs at basal state, in the absence of Ca(2+)/CaM, and is inversely correlated with substrate phosphorylation. This type of phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C(6)-ceramide. The substitution of Ser(308) to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca(2+)/CaM-independent substrate phosphorylation as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. Conversely, mutation to aspartic acid reduces the binding of the protein to CaM and abrogates almost completely the death-promoting function of the protein. These results are consistent with a molecular model in which phosphorylation on Ser(308) stabilizes a locked conformation of the CaM-regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device, which keeps death-associated protein kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.
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PMID:The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. 1157 85

We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.
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PMID:PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites. 1179 24


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