3-Phosphoglycerate-dependent protein phosphorylation

The EMBO Journal, 1997

number of possible second messenger roles for Ins(1,3,4,5)P 4 , e.g. in Ca 2ϩ homeostasis (Changya et al., D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] 3-kin-1989; Irvine, 1991). Moreover, the recent cloning and ase, the enzyme responsible for production of D-myocharacterization of a specific high affinity Ins(1,3,4,5)P 4 inositol 1,3,4,5-tetrakisphosphate, was activated 3-to binding protein from pig platelets demonstrated that it 5-fold in homogenates of rat brain cortical slices after corresponds to a member of the GTPase activating protein incubation with carbachol. The effect was reproduced (GAP) 1 family (Ras-GAP1 IP4BP). This protein showed in response to UTP in Chinese hamster ovary (CHO) GAP activity towards Ras and was specifically stimulated cells overexpressing Ins(1,4,5)P 3 3-kinase A, the major by Ins(1,3,4,5)P 4 (Cullen et al., 1995). isoform present in rat and human neuronal cells. In cDNAs encoding rat and human brain Ins(1,4,5)P 3 3ortho-32 P-labelled cells, the phosphorylated 53 kDa kinase A (50-53 kDa) have been isolated (Choi et al., enzyme could be identified after receptor activation by 1990; Takazawa et al., 1990b, 1991a). Evidence has been immunoprecipitation. The time course of phosphorylaprovided to show high expression of isoform A in neuronal tion was very similar to that observed for carbachol cells of the cortex, hippocampus and cerebellum in both (or UTP)-induced enzyme activation. Enzyme phosphorat and human (Mailleux et al., 1991, 1992). Rat and rylation was prevented in the presence of okadaic acid. human Ins(1,4,5)P 3 3-kinases A show 93% amino acid Calmodulin (CaM) kinase II inhibitors (i.e. KN-93 and sequence identity and polyclonal antibodies to the purified KN-62) prevented phosphorylation of Ins(1,4,5)P 3 3rat brain isoenzyme A also recognize the human isoform kinase. Identification of the phosphorylation site in A (Takazawa et al., 1991a). cDNAs encoding an isotransfected CHO cells indicated that the phosphorylenzyme, i.e. Ins(1,4,5)P 3 3-kinase B, have been isolated ated residue was Thr311. This residue of the human from human and rat cDNA libraries (Takazawa et al., brain sequence lies in an active site peptide segment 1991b; Thomas et al., 1994; Vanweyenberg et al., 1995). corresponding to a CaM kinase II-mediated phospho-Both indirect and direct evidence suggests that rylation consensus site, i.e. Arg-Ala-Val-Thr. The same Ins(1,4,5)P 3 3-kinase is controlled by various mechanisms. residue in Ins(1,4,5)P 3 3-kinase A was also phosphoryl-Ca 2ϩ regulates Ins(1,3,4,5)P 4 production in lysed thymoated in vitro by CaM kinase II. Phosphorylation cytes and in intact cells stimulated with concanavalin A resulted in 8-to 10-fold enzyme activation and a 25-(Zilberman et al., 1987). In the lacrimal acinar cells, fold increase in sensitivity to the Ca 2ϩ :CaM complex. acetylcholine activates Ca 2ϩ-dependent K ϩ channels even In this study, direct evidence is provided for a novel when Ins(1,4,5)P 3 perfused into the same cells does not regulation mechanism for Ins(1,4,5)P 3 3-kinase (iso-(Morris et al., 1987). It was suggested that acetylcholine form A) in vitro and in intact cells. promotes the production of Ins(1,3,4,5)P 4 and that possibly Keywords: calcium/calmodulin/inositol phosphate/kinase/ Ins(1,4,5)P 3 3-kinase is stimulated by receptor activation phosphorylation (Irvine et al., 1988). Purified Ins(1,4,5)P 3 3-kinase appeared to be sensitive to the Ca 2ϩ :calmodulin (CaM) complex, 2-fold in rat and human brain (Lee et al., 1990;

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998

The plasma membrane Ca 2 -ATPase (PMCA), the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several independent mechanisms. In this paper we report that the protein kinases A and C differentially activate the Ca 2 -ATPase purified from synaptosomal membranes of rat cortex, cerebellum and hippocampus. The effect of protein kinases was more pronounced for the cortical enzyme, whereas cerebellar and hippocampal Ca 2 -ATPases were activated to a lesser degree. The preparation of Ca 2 -ATPase contained the phosphoamino acids, i.e., P-Ser and P-Thr, indicating that the enzyme was purified in phosphorylated state. The phosphorylation of Ca 2 -ATPase by PKA and PKC increased the amount of phosphoamino acids, but in a region-dependent manner. Using the specific antibodies against N-terminal portion of four main PMCA isoforms we have characterized the isoforms composition of Ca 2 -ATPase purified from the nervous endings of examined brain areas. Our results indicate that the activity of calcium pump is related to its phosphorylated state, and that the phosphorylation is region-dependent. Moreover, the differences observed could be related to the composition of PMCA isoforms in the different brain areas. Phosphorylation of the plasma membrane Ca 2 -ATPase appears to be a mechanism to control its activity. The results support also the possible involvement of PKA and PKC. ß

Journal of Biological Chemistry, 1999

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (K m ‫؍‬ 0.2 M) over cAMP-dependent protein kinase (K m ‫؍‬ 2.0 M). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC 50 of 131 ؎ 27 nM. Dephospho-G-substrate was not found to be inhibitory. Both dephospho-and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A 1 , which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases. Cyclic GMP acts as a second messenger in numerous physiological processes including smooth muscle relaxation, visual transduction, olfaction, neurotransmitter release, and long term depression of cerebellar Purkinje cells (1-3). cGMP medi

Journal of Neurochemistry, 2002

Activation of phospholipase D (PLD) is involved in receptor-mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PAregulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain-specific 25-kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti-p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an antiprotein kinase C (PKC)ct antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA-and PSstimulated PARK activity was extremely augmented by the presence of 1~uMfree Ca 2~.In the presence of 1 mM EGTA, phorbol 1 2-myristate 13-acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCa. From these results, it is suggested that the PARK activity purified might be attributed to PKCa. In p25-depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti-p25 antibody, PA-dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCa in bovine brain cytosol was depleted by its precipitation with the anti-PKCa antibody, neither p29 nor p32 in this PKCa-depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCa, which, in turn, phosphorylates p29, which may be identical with p25.

Molecular Brain Research, 1996

A full-length cDNA for a novel isoform of the human receptor tyrosine phosphatase y gene d'I l'I«,\ was overexpressed in Sf9 insect cells, and the gene product. PTPy, was purified and characterized. The protein was expressed as a Mr~185,000 protein accompanied by a Mr â€"¿ 120,000 putative cleavage product on SDS-PAGE analysis. The protein undergoes jV-linked glycosylation and constitutive phosphorylation of serine resi dues. When assayed for tyrosine-specific phosphatase activity, PTPy dephosphorylatcd myelin basic protein at a pH optimum of 7.5 and a A,„ of 12.6 UM; reduced carboxyamidomethylated and maleylated lysozyme (RCM-lysozyme) at a pH optimum of 6.0 and a k,,, of 12 UM; and p-nitrophenylphosphate with a pH optimum of 5.5 and a A,,, of 3.5 HIM.

Molecular & Cellular Proteomics, 2006

1 The abbreviations used are: CaMKII, Ca 2ϩ /calmodulin-dependent protein kinase II; NMDA, N-methyl-D-aspartate; SCX, strong cation exchange chromatography; PSD, postsynaptic density; iTRAQ, isobaric peptide tags for relative and absolute quantification; GO, gene ontology; DAP, disks large-associated protein; PKC, protein kinase C.

Archives of Biochemistry and Biophysics, 1987

The Journal of Biological Chemistry, 1986

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac CAMPdependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction ( K , = 20 p~; V,,, = 2 pmol min" mg") are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S . aureus VS protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RE. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99) was dephosphorylated with kinetics comparable to R I~ ( K , = 26 p~, Vmax= 1.7 pmol min-l mg"). Structural analysis of this peptide indicates that an amphipathic &sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.