GTP-binding protein gamma12 subunit phosphorylation by protein kinase C--identification of the phosphorylation site and factors involved in cultured cells and rat tissues in vivo

We have demonstrated previously that the GTP-binding protein gamma12 subunit is a selective substrate for phosphorylation by protein kinase C among various gamma subunits in vitro, and that a serine residue in the N-terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N-terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated gamma12 and Swiss 3T3 cells rich in this protein, gamma12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12-myristate 13-acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of gamma12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of G(i/o). Phosphorylation of gamma12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet-derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP-binding proteins and protein kinase C in combination. Phosphorylated gamma12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of G(i/o) in vivo.     

Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.     

The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259

The kinase negative aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein was phosphorylated in vitro after treatment with lysates from A431 cells and subsequent in vitro protein kinase assay. This phosphorylation was independent of stimulation of the cells with EGF; it occurred exclusively on serine and was mapped to Ser259. The identical site of c-Raf-1 was phosphorylated in A431 cells by metabolic labelling in vivo. The kinase binding domain was mapped by various GST-Raf deletion mutants to c-Raf-1 aminoacid residues 181 to 255.     

HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.     

Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.     

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.     

Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin

Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km approximately tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361-Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above-mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.     

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.     

Spatial patterns of Ca2+ signals define intracellular distribution of a signaling by Ca2+/Calmodulin-dependent protein kinase II

Ca2+ plays a central role in cell signaling, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+ actions. The spatial distribution of intracellular Ca2+ signaling is not homogenous, rather it is dynamically organized, and it has been speculated that spatial patterns of Ca2+ signals may function as a form of cellular information transmitted to downstream molecules. To address this issue, we studied the intracellular distributions of the signalings by CaMKII and Ca2+ in the same astrocytes. The former was visualized by monitoring site-specific phosphorylation of a cytoskeletal protein vimentin, using site- and phosphorylation-specific antibodies, while the latter was examined by fura-2-based Ca2+ microscopy. Local Ca2+ signals induced vimentin phosphorylation by CaMKII localized in the same area. On the other hand, Ca2+ waves in astrocytes induced global phosphorylation of vimentin by CaMKII. A small population of vimentin filaments highly phosphorylated by CaMKII underwent structural alteration into short filaments at electron microscopic level. These results indicate that CaMKII transmits spatial patterns of Ca2+ signals to vimentin as cellular information. The possibility is discussed that spatial patterns of vimentin phosphorylation may be important for intracellular organization of vimentin filament networks.     

Topography of the interaction of HPr(Ser) kinase with HPr

The phosphocarrier protein, HPr, from Gram-positive organisms and mycoplasmas is a substrate for an ATP-dependent kinase that phosphorylates serine 46. In Gram-negative organisms, the corresponding HPr is not phosphorylated on serine 46 and the ATP-dependent kinase is absent. To determine the specificity requirements for phosphorylation of Mycoplasma capricolum HPr, a chimera in which residues 43-57 were replaced by the Escherichia coli sequence was constructed. The chimeric protein folded properly, but was not phosphorylated on either serine 46 or histidine 15. A dissection of the region required for phosphorylation specificity was carried out by further mutagenesis. The deficiency in phosphorylation at histidine 15 was localized primarily to the region including residues 51-57. Activity studies revealed that residues 48, 49, and 51-53 are important for recognition of M. capricolum HPr by its cognate HPr(Ser) kinase. The characteristics of this region suggest that the kinase-HPr interaction occurs mainly through a hydrophobic region. Molecular modeling comparisons of M. capricolum HPr and the chimeric construct provided a basis for interpreting the results of the activity assays.     

Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.     

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.     

Sodium lactate affects sensory and objective characteristics of tray-packed broiler chicken breast meat

We have determined the minimal portion of the retinoblastoma protein (Rb) that can serve as an efficient substrate for in vitro phosphorylation by cdk4 kinase-D1 cyclin. Kinetic measurements indicate that in vitro, a 15-kDa fragment that represents the C-terminus of Rb can serve equally well as a substrate when compared with the larger 56-kDa fragment of Rb, which contains the A, B and C domains. By comparison, peptide substrates appear to be 1000-fold less efficient. Furthermore, mutational analysis indicates that not all of the five phosphorylation sites within this minimal C domain are phosphorylated equally by cdk4/D1. Ser795 is the preferred phosphorylation site, whereas the four remaining sites Ser807, Ser811, Thr821 and Thr826 are phosphorylated to a much lesser degree. Truncations of the C domain from the carboxy terminus indicate that almost all of this domain is required for efficient phosphorylation. These data suggest that the structural context of the phosphorylation site within the substrate is critical for its phosphorylation by the cdk4/D1 kinase.     

cAMP-dependent phosphorylation of two sites in the alpha subunit of the cardiac sodium channel

The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.     

A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.     

Studies on the phosphorylation of a M(r) 25,000 protein, a putative protein phosphatase 2A modulator, by casein kinase I, and analysis of multiple endogenous phosphates

A M(r) 25,000 protein, which was isolated from the cytosolic fraction of Xenopus laevis oocytes, is a newly identified substrate for casein kinase II and protein kinase C [Hashimoto et al. (1995) J. Biochem. 118, 453-460], and was recently shown to have the ability to modulate protein phosphatase 2A activity [Hashimoto et al. (1996) J. Biochem. 119, 626-632]. Acid phosphatase treatment of the protein shifted its electrophoretic mobility from 25 to 20 kDa on SDS-PAGE. The content of alkali-labile phosphate bound covalently to the protein was 53 mol per mol of M(r) 25,000 protein. Amino acid composition analysis revealed that there are 50 serine residues and 6 threonine residues per mol of this protein. Therefore, this M(r) 25,000 protein seems to be highly phosphorylated in vivo. The M(r) 25,000 protein, once partially dephosphorylated by acid phosphatase, served as an efficient substrate for casein kinase I and casein kinase II. When entirely dephosphorylated, the M(r) 25,000 protein was used as a substrate, the rate of phosphorylation with both casein kinases being decreased. This behavior of casein kinases toward the M(r) 25,000 protein reflects the possible mechanism of multisite phosphorylation in which the introduction of a phosphate group facilitates sequential phosphorylation.     

Protein kinase C-dependent in vivo phosphorylation of prourokinase leads to the formation of a receptor competitive antagonist

We recently reported that in vivo phosphorylation of urokinase-type plasminogen activator on Ser138/303 prevents its catalytic-independent ability to promote myelomonocytic cell adherence and motility. We now show that Ca2+ activated, phospholipid-dependent protein kinase C from rat brain phosphorylates in vitro a peptide corresponding to prourokinase residues 133-143 (DGKKPSSPPEE) and the full-length molecule on Ser138/139. The in vivo involvement of the protein kinase C isoenzyme family is supported by the finding that inhibition of kinase C activity prevents prourokinase phosphorylation on Ser138/303 in A431 human carcinoma cells. Conversely, a short treatment of A431 cells with phorbol myristate acetate increases the extent of phosphorylated prourokinase and, concomitantly, affects its function; under these conditions, the capability of prourokinase to up-regulate U937 monocyte-like cell adherence is severely impaired, although receptor binding is unaltered. By the aid of a "phosphorylation-like" variant (Ser138 to Glu) we show that modification of Ser138 is sufficient to confer to prourokinase the antagonistic properties observed following in vivo stimulation of protein kinase C activity. These observations provide the first evidence that protein kinase C directs the formation of a receptor competitive antagonist by regulating the in vivo phosphorylation state of prourokinase.     

Steady-state plasma membrane expression of human cytomegalovirus gB is determined by the phosphorylation state of Ser900

Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser900. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser900, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser900 was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser900 trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the trans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.     

Identification and partial characterization of factor Va heavy chain kinase from human platelets

Factor Va, the essential cofactor for prothrombinase, is phosphorylated on the acidic COOH terminus of the heavy chain of the cofactor, at Ser692, by a platelet membrane-associated casein kinase II (CKII). Consistent with this observation, phosphorylation of the factor Va heavy chain by the platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was monitored using a polyclonal antibody to the alpha-subunit, and kinase activity in the various fractions was confirmed using human factor Va as a substrate. Immunoblotting experiments using polyclonal antibodies raised against synthetic peptides mimicking a portion of the deduced amino acid sequence of the alpha-, alpha'-, and beta-subunits of human CKII demonstrated the coexistence of both alpha- and alpha'-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha'beta2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alphaalpha/betabeta and one that is alpha'alpha'/betabeta. In the presence of the purified platelet-derived CKII, human factor Va incorporates between 0.8 and 1.3 mol of phosphate/mol of factor Va depending on the concentration of the beta-subunit in the kinase preparation. A peptide mimicking the sequence 687-705 of the human factor V molecule incorporates radioactivity in the presence of purified CKII and inhibits factor Va heavy chain phosphorylation by the platelet CKII. In contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation by the platelet CKII. Human factor Va is inactivated by activated protein C following three cleavages of the heavy chain at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure of the inactivating cleavage site at Arg306. The phosphorylated cofactor has increased susceptibility to inactivation by activated protein C, since phosphorylated factor Va was found to be inactivated approximately 3-fold faster than its native counterpart. Acceleration of the inactivation process of the phosphorylated cofactor occurs because of acceleration of the rate of cleavage at Arg506. These data suggest a critical role for factor Va phosphorylation on the surface of platelets in regulating cofactor activity.     

Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear targeting

Translocation of protein kinases with broad substrate specificities between different subcellular compartments by activation of signaling pathways is an established mechanism to direct the activity of these enzymes toward particular substrates. Recently, we identified two isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), which are targeted to the nucleus by an alternatively spliced nuclear localization signal (NLS). Here we report that cotransfection with constitutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylation of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation is both necessary and sufficient to block nuclear targeting. Furthermore, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces binding of the kinase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that we have observed in situ.