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)

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.
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PMID:Alternative binding modes of an inhibitor to two different kinases. 1286 92

The HePTP (haematopoietic protein tyrosine phosphatase) is a negative regulator of the ERK2 (extracellular signal-regulated protein kinase 2) and p38 MAP kinases (mitogen-activated protein kinases) in T-cells. This inhibitory function requires a physical association of HePTP through an N-terminal KIM (kinase-interaction motif) with ERK and p38. We previously reported that PKA (cAMP-dependent protein kinase) phosphorylates Ser-23 within the KIM of HePTP, resulting in dissociation of HePTP from ERK2. Here we follow the phosphorylation of this site in intact T-cells. We find that HePTP is phosphorylated at Ser-23 in resting T-cells and that this phosphorylation increases upon treatment of the cells with agents that elevate intracellular cAMP, such as prostaglandin E2. HePTP phosphorylation occurred at discrete regions at the cell surface. Phosphorylation was reduced by inhibitors of PKA and increased by inhibitors of protein phosphatases PP1 and PP2A, but not by inhibitors of calcineurin. In vitro, PP1 efficiently dephosphorylated HePTP at Ser-23, while PP2A was much less efficient. Activation of PP1 by treatment of the cells with ceramide suppressed Ser-23 phosphorylation, as did transfection of the catalytic subunit of PP1. Phosphorylation at Ser-23 is also increased in a transient manner upon T-cell antigen receptor ligation. In contrast, treatment of cells with phorbol ester had no effect on HePTP phosphorylation at Ser-23. We conclude from these results that HePTP is under continuous control by PKA and a serine-specific phosphatase, probably PP1, in T-cells and that this basal phosphorylation at Ser-23 can rapidly change in response to external stimuli. This, in turn, will affect the ability of HePTP to inhibit the ERK and p38 MAP kinases.
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PMID:Haematopoietic protein tyrosine phosphatase (HePTP) phosphorylation by cAMP-dependent protein kinase in T-cells: dynamics and subcellular location. 1461 83

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa. At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial alpha-crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein. At pH 6.0, both alpha-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, alpha-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase. The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperature-dependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.
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PMID:Some properties of human small heat shock protein Hsp20 (HspB6). 1471 97

Three mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2, MK2) interacting proteins were identified using a yeast two-hybrid approach. ShcA, a signaling phospho-protein, human polyhomeotic 2 (HPH2), a transcriptional regulator, and highly similar to smoothelin (HSTS), which is related to the cytoskeletal associated protein smoothelin, interact specifically with MK2. The interaction of MK2 with the 66 kDa isoform of ShcA, p66(ShcA), and HPH2 was confirmed using co-immunoprecipitation. MK2 is activated with p66(ShcA) co-expression and p66(ShcA) is an in vitro substrate for MK2, further demonstrating their association and suggesting a biological role for p66(Shc) in MK2 activation.
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PMID:P66(ShcA) interacts with MAPKAP kinase 2 and regulates its activity. 1509 67

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.
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PMID:Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter. 1521 32

Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.
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PMID:Nek2A specifies the centrosomal localization of Erk2. 1535 3

Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Additionally, transgenic plants with CaMV35S promoter::SRK2C-GFP displayed higher overall drought tolerance than control plants. Whereas stomatal regulation in 35S::SRK2C-GFP plants was not altered, microarray analysis revealed that their drought tolerance coincided with up-regulation of many stress-responsive genes, for example, RD29A, COR15A, and DREB1A/CBF3. From these results, we concluded that SRK2C is capable of mediating signals initiated during drought stress, resulting in appropriate gene expression. Our present study reveals new insights around signal output from osmotic-stress-activated SnRK2 protein kinase as well as supporting feasibility of manipulating SnRK2 toward improving plant osmotic-stress tolerance.
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PMID:SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. 1556 75

The neurofibromatosis 2 tumour suppressor merlin/schwannomin is structurally related to the ezrin-radixin-moesin family of proteins, which anchor actin cytoskeleton to specific membrane proteins and participate in cell signalling. Merlin inhibits cell growth with a yet unknown mechanism. As most tumour suppressors are linked to cell cycle control, we investigated merlin's behaviour during cell cycle. In glioma and osteosarcoma cells, endogenous merlin was targeted to the nucleus in a cell cycle-specific manner. Merlin accumulated perinuclearly at the G2/M phase, and shifted to the nucleus at early G1. During mitosis, merlin localized to mitotic spindles and at the contractile ring. Nuclear merlin was strongly reduced in confluent cells. Blocking of the CRM1/exportin nuclear export pathway led to accumulation of merlin in the nucleus. Activation of the p21-activated kinase or protein kinase A, which result in phosphorylation of merlin, did not affect its nuclear localization. Merlin regulates the activity of extracellular signal-regulated kinase 2 (ERK2) and nuclear localization of both proteins was induced by cell adhesion. Unlike ERK2, nuclear localization of merlin was not, however, dependent on intact actin cytoskeleton. These results link merlin to events related to cell cycle control and may help to resolve its tumour suppressor function.
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PMID:Cell cycle-dependent nucleocytoplasmic shuttling of the neurofibromatosis 2 tumour suppressor merlin. 1558 Feb 88

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.
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PMID:A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates. 1578 14

A substrate for PKBalpha (protein kinase Balpha) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBalpha, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser52 in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser58). CRHSP24 became phosphorylated at Ser52 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1[L155E]) that activates PKBalpha normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser52 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBalpha, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser30, Ser32 and Ser41 in vitro, and Ser41 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser52 by PKBalpha in response to IGF-1, at Ser52 by PKBalpha and RSK in response to EGF, and at Ser41 in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).
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PMID:Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL. 1591 Feb 84


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