Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.
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PMID:Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle. 20 8

Filamin is a high molecular weight protein that binds to actin filaments in cells. It is found in large amounts in several different cells and tissues, including smooth muscle, fibroblasts, platelets, and macrophages. It is immunologically related to the previously described macrophage high molecular weight actin-binding protein but clearly different from erythrocyte spectrin. Filamin is a phosphoprotein; it is phosphorylated in vivo in intact tissues and cells. It can be phosphorylated in vitro with endogenous kinases; cyclic AMP stimulates this phosohorylation. Furthermore, the purified protein can be phosphorylated by purified cyclic AMP-dependent protein kinase. In smooth muscle homogenates, the stimulation of filamin phosphorylation by cyclic AMP is specific. Cyclic GMP and Ca2+ do not increase its phosphorylation, although they do stimulate phosphorylation of other proteins.
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PMID:Cyclic AMP-dependent phosphorylation of the actin-binding protein filamin. 20 86

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.
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PMID:Purification of mammalian filamin. Similarity to high molecular weight actin-binding protein in macrophages, platelets, fibroblasts, and other tissues. 64 Oct 65

We have constructed a galactose-inducible expression library by cloning yeast cDNAs unidirectionally under control of the GAL1 promoter in a centromeric shuttle vector. Eleven independent libraries were made each with an average size of about 1 x 10(6) clones, about 50 times larger than the reported mRNA population in a yeast cell. From this library, LEU2 and HIS3 cDNAs were recovered at a frequency of about 1 in 10(4) and in 12 out of 13 cases these were expressed in a galactose-dependent manner. Sequence analysis of leu2 and his3 complementing cDNAs indicates that they contain all the coding sequence and much of the 5' untranslated region. To test the utility of the library for the identification of genes whose overexpression confers a specific phenotype, we screened 25,000 yeast transformants for lethality on galactose. Among 15 clones that showed galactose inducible lethality were cDNAs encoding structural proteins, including ACT1 (actin), TUB2 (beta-tubulin) and ABP1 (actin-binding protein 1), and genes in signal transduction pathways, including TPK1 (a cAMP-dependent protein kinase) and GLC7 (type 1 protein phosphatase). cDNAs overexpressing NHPB (nonhistone protein B) and NSR1 (nuclear sequence recognition protein) were also found to be lethal. Among these, ACT1 was isolated four times, and NSR1 three times. The useful features of this library for cDNA cloning in yeast by complementation, and for the identification of genes whose over-expression confers specific phenotypes, are discussed.
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PMID:Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. 146 25

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.
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PMID:Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the cAMP-dependent protein kinase in bovine platelets. 173 23

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.
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PMID:Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C. 205 Jun 83

A question of whether or not casein kinase II (CKII) activity associated with Fc gamma 2aR is involved in the regulation of phagocytic process mediated by this type of Fc gamma R was investigated. Our previous studies showed that the rate of phagocytosis of sheep erythrocytes (SRBC) coated with anti-SRBC antibody (EA) by P388D1 cells varies significantly depending on the isotypes of antibody and that Fc gamma 2aR isolated from the detergent lysate of P388D1 cells is associated with CKII activity, whereas Fc gamma 2bR is not. Fc gamma Rs-mediated phagocytosis is a major function of macrophages by which invading pathogens such as bacteria could be eliminated and therefore warrants the investigation of its biochemical mechanisms. We have recently shown that phagocytosis of EA2b mediated by Fc gamma 2bR of P388D1 cells as well as murine peritoneal macrophages could be up-regulated by promoting the association of various cytoskeletal components with the receptor by inhibiting Fc gamma 2bR-associated phospholipase A2 (PLA2). CKII activity-associated Fc gamma 2aR mediates phagocytosis of EA2a more effectively than PLA2-associated Fc gamma 2bR mediates phagocytosis of EA2b. We have therefore examined a potential role of CKII in Fc gamma 2aR-mediated phagocytosis by the use of a specific inhibitor of CKII activity (heparin). Results showed that heparin inhibited CKII activity associated with Fc gamma 2aR and effectively down-regulated the Fc gamma 2aR-mediated phagocytosis by apparently blocking the association of the receptor with four types of cytoskeletal components (actin-binding protein, myosin heavy chain, alpha tubulin, and actin).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of Fc gamma 2a receptor-mediated phagocytosis by a murine macrophage-like cell line, P388D1: involvement of casein kinase II activity associated with Fc gamma 2a receptor. 253 85

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
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PMID:In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. 254 93

Actomyosin in smooth muscle is in a quiescent state. The mechanism or mechanisms by which Ca2+ activates the actomyosin ATPase is not clear. There is sufficient evidence for the presence of enzyme systems which phosphorylate and dephosphorylate myosin light chains. The activity of the kinase that phosphorylates the myosin is regulated by cAMP-dependent protein kinase. Phosphorylated kinase has decreased affinity for calmodulin and lower activity when compared with unphosphorylated myosin light chain kinase. The activity of myosin light chain kinase is also regulated by calcium-calmodulin. In the presence of Ca2+, myosin is phosphorylated. In the absence of Ca2+, the phosphatase activity becomes dominant; the myosin remains in the unphosphorylated form under this condition. The Mg2+-ATPase of the phosphorylated myosin is activated by actin. The maximal activation of the Mg2+-ATPase by actin requires Ca2+ and tropomyosin, a protein located on the thin filament. Hence, the actin-activation of the Mg2+-ATPase requires Ca2+ even after phosphorylation by the calcium-calmodulin dependent kinase. The regulation of actin-activated ATPase activity by myosin light chain phosphorylation is depicted in the schematic diagram. Caldesmon, an actin-binding protein which also binds to calmodulin in the presence of Ca2+, has been shown to be present in thin-filaments isolated from smooth muscle. This protein inhibits actin-activated myosin ATPase activity. The release from this inhibition requires Ca2+ and calmodulin. The possibility that caldesmon is also involved in the calcium regulation of actomyosin in smooth muscle is presently under investigation in a number of laboratories.
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PMID:Regulation of actomyosin ATPase in smooth muscle. 294 44

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein in unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A2, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, though other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.
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PMID:Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs. 679 1


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