Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes

A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.     

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.     

Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

Primary structure and domain organization of human alpha and beta adducin

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH- terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH- termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH- terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.     

AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.     

Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.     

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.     

Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro

We have previously reported that fodrin (beta subunit), tubulin (alpha subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase (Kadowaki, T., Nishida, E., Kasuga, M., Akiyama, T., Takaku, F., Ishikawa, M., Sakai, H., Kathuria, S., and Fujita-Yamaguchi, Y. (1985) Biochem. Biophys. Res. Commun. 127, 493-500 and Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S., Akanuma, Y., and Kasuga, M. (1985) J. Biol. Chem. 260, 4016-4020). In this study, to investigate the substrate specificities of tyrosine kinases, we have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the beta subunit, but not the alpha subunit, of fodrin on tyrosine residues with a Km below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the alpha subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP2 was the best substrate for the EGF receptor kinase. By contrast, src kinase favored phosphorylation of tubulin as compared to MAP2. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred beta subunit to alpha subunit, but the src kinase phosphorylated both alpha and beta subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.     

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.     

Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation

The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649-6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP-dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits.     

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differing modulation of protein kinase C by bryostatin 1 and phorbol esters in JB6 mouse epidermal cells

Bryostatin 1 (Bryo), a macrocyclic lactone, stimulates some but not all of the biologic effects which are induced by phorbol esters (PEs). In vitro, it competes with PEs for binding to whole cells and activates the calcium/phospholipid-dependent protein kinase, PK-C. To examine whether Bryo, like PEs, is able to stimulate the nonadherent growth of cells, we used the mouse epidermal cell line JB6, which is stimulated by PEs to grow in soft agar. Like PEs, Bryo stimulates both the adherent and nonadherent growth of these cells, but Bryo (0.001-1 microM) is less active than equivalent concentrations of PEs. To attempt to explain the biologic differences between these two agents, we examined the modulation of PK-C by both PEs and Bryo. In a phosphotransferase assay using partially purified PK-C from JB6 cells, Bryo (1-0.001 microM) stimulated less phosphorylation of histone substrate than did PMA. Also, when whole cells were treated with equal concentrations of Bryo or PMA, Bryo stimulated a decreased loss of PK-C from the cytosol. Using purified isozymes of PK-C from rat brain, Bryo demonstrated identical competition to PMA for binding to forms alpha and gamma but decreased binding to form beta. Hydroxylapatite chromatography of JB6 cytosol demonstrated that these cells contain largely peak 2, or beta-PK-C. Although Bryo more weakly activates PK-C from JB6 cells, prolonged exposure of JB6 cells to either 1.0 or 0.01 microM Bryo caused a more rapid loss of immunologically detectable PK-C than did similar concentrations of PEs. We conclude that Bryo is capable of stimulating both the nonadherent and the adherent growth of JB6 cells in a similar fashion to phorbol esters. The differences in biologic effects of Bryo and PMA may be partially explained by Bryo's modulation of PK-C.     

Cyclic AMP-dependent protein kinase (PKA) phosphorylates Ser362 and 467 and protein kinase C phosphorylates Ser550 within the M3/M4 cytoplasmic domain of human nicotinic receptor alpha4 subunits

Studies have suggested that the expression, translocation, and function of alpha4beta2 nicotinic receptors may be modulated by alpha4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on alpha4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human alpha4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type beta2 subunits into SH-EP1 cells. Cells were pre-incubated with (32)Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated alpha4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two alpha4 protein bands, a major band of 71/75 kDa and a minor band of 80/85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the alpha4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.     

Phosphorylation of casein kinase II

Casein kinase II from rabbit reticulocytes is a tetramer with an alpha,alpha' beta 2 or alpha 2 beta 2 structure; the alpha subunits contain the catalytic activity, and the beta subunits are regulatory in nature [Traugh, J.A., Lin, W. J., Takada-Axelrod, F., & Tuazon, P. T. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 224-229]. When casein kinase II is isolated from rabbit reticulocytes by a rapid two-step purification of the enzyme, both the alpha and beta subunits are phosphorylated to a significant extent. In vitro, purified casein kinase II undergoes autophosphorylation on the beta subunit. In the presence of polylysine and polyarginine, phosphorylation of the beta subunits is inhibited, and the alpha subunits (alpha and alpha') become autophosphorylated. The effectiveness of polylysine coincides with the molecular weight. With basic proteins, including a number of histones and protamine, autophosphorylation of both subunits is observed. With histones, autophosphorylation of each subunit can be greater than that observed with the autophosphorylated enzyme alone or with a basic polypeptide. Thus, the potential exists for modulatory proteins to alter the autophosphorylation state of casein kinase II. Taken together, the data suggest that phosphorylation of the alpha subunit of casein kinase II in vivo may be due to an unidentified protein kinase or due to autophosphorylation. In the latter instance, casein kinase II could be transiently associated with specific intracellular compounds, such as basic proteins, with a resultant stimulation of autophosphorylation.     

Endogenous modulators of glucocorticoid receptor function also regulate purified protein kinase C

Modulator-1 and -2, proposed to be novel ether-linked aminophosphoglycerides, were originally identified as regulators of glucocorticoid receptor function (Bodine, P. V., and Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554). We now demonstrate that these modulators are also potent new stimulators of protein kinase C activity in vitro. These endogenous biomolecules regulate purified protein kinase C activity in a biphasic and dose-dependent pattern, as determined by histone phosphorylation. Modulators, at concentrations within their apparent cellular range, stimulate protein kinase C-catalyzed histone phosphorylation 2-4-fold when added separately, or 10-12-fold when added together. This enhancement of kinase activity apparently is specific for protein kinase C, since neither protein kinase M, nor cAMP-dependent protein kinase A are stimulated by the modulators. The stimulation of purified protein kinase C occurs only when the enzyme has been initially activated by calcium, phosphatidylserine, and diacylglycerol, indicating that the modulators do not simply substitute for one of the enzyme cofactors. In addition, the modulators appear to interact directly with protein kinase C, perhaps with the regulatory domain of the enzyme, since these biomolecules inhibit the binding of phorbol ester to purified protein kinase C. Finally, time-course studies of protein kinase C-catalyzed histone phosphorylation indicate that the velocity of the enzyme reaction is increased by the modulators. Taken together, these results suggest that the modulators are a new class of regulators of protein kinase C.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)