Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils

A series of 8 peptides derived from the amino acid sequence accommodating the plasmin cleavage site in vitronectin were synthesized and used to map its binding site for the type I plasminogen activator inhibitor (PAI-1). This mapping assigned the inhibitor binding site to the K348-R370 region with high affinity recognition elements within the K348-R357 sequence. These results account for our previous finding that cleavage of the R361-S362 bond by plasmin significantly reduces the affinity between PAI-1 and vitronectin, since it splits the PAI-1 binding site in two. Furthermore, in the case of the two-chain form of vitronectin, this cleavage detaches the S362-R379 peptide which provides some of the affinity elements for the binding of PAI-1.     

Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms

The majority of familial Alzheimer's disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N-terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its approximately 20 kDa CTF involving protein kinase C (PKC) and/or protein kinase A (PKA). We now have found that the CTF of the highly homologous PS-2 is also phosphorylated. Surprisingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, the PS-2 CTF is constitutively phosphorylated in vivo by serine/threonine protein kinases, which are independent of phorbol ester and intracellular cAMP. In vitro the large hydrophilic loop of PS-2 between transmembrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK-1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed that the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosphorylate the CTF of PS-2 within its hydrophilic loop domain in vivo. Interestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.     

Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits. Yet, the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.     

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.     

Regulation of N-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.     

Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase

It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.     

Vitronectin expression in rheumatoid arthritic synovia--inhibition of plasmin generation by vitronectin produced in vitro

The plasmin-generating system controls, to a great extent, the degree of connective tissue destruction as well as fibrin deposition two contributors to the pathogenesis generated in diseases such as rheumatoid arthritis. Vitronectin, an adhesive blood glycoprotein, has the potential to modulate this system by its known capacity to interact with plasminogen activator inhibitor-1, plasminogen activators, the urokinase plasminogen activator receptor, and plasminogen. The net effect of these interactions, in terms of plasmin generation, is not known as yet. In the present study, we have investigated the possible expression and role of vitronectin in rheumatoid arthritic synovia. Analysis of synovial frozen sections by immunofluorescence showed the presence of vitronectin in the 13 cases studied. In situ hybridization analysis demonstrated the presence of vitronectin mRNA in cells present in areas rich in infiltrating inflammatory cells. The adherent population of the rheumatoid arthritic synovial cells was isolated and found to synthesize and secrete vitronectin into the medium (seven of 10 isolates), as assessed by metabolic labelling and immunoprecipitation. Plasmin-generating activity was detected in the adherent synovial cells, and this activity was increased by antibodies to vitronectin. Our findings show, for the first time, that vitronectin can be endogenously produced in a pathophysiological system where it can inhibit the generation of plasmin.     

Estrogen improves biochemical and neurologic outcome following traumatic brain injury in male rats, but not in females

Previous work has shown that the GABAA-receptor (GABAA-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit/subtypes of the GABAA-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the beta 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the beta 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315-1317) that both PKA and PKC phosphorylate the beta-subunit of the GABAA-R.     

Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.     

Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants

The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.     

Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein by using mouse monoclonal antibodies

BACKGROUND: Bacterial resistance to aminoglycoside antibiotics occurs primarily through the expression of modifying enzymes that covalently alter the drugs by O-phosphorylation, O-adenylation or N-acetylation. Aminoglycoside phosphotransferases (APHs) catalyze the ATP-dependent phosphorylation of these antibiotics. Two particular enzymes in this class, APH(3')-IIIa and AAC(6')-APH(2"), are produced in gram-positive cocci and have been shown to phosphorylate aminoglycosides on their 3' and 2" hydroxyl groups, respectively. The three-dimensional structure of APH (3')-IIIa is strikingly similar to those of eukaryotic protein kinases (EPKs), and the observation, reported previously, that APH(3')-IIIa and AAC(6')-APH(2") are effectively inhibited by EPK inhibitors suggested the possibility that these aminoglycoside kinases might phosphorylate EPK substrates. RESULTS: Our data demonstrate unequivocally that APHs can phosphorylate several EPK substrates and that this phosphorylation occurs exclusively on serine residues. Phosphorylation of Ser/Thr protein kinase substrates by APHs was considerably slower than phosphorylation of aminoglycosides under identical assay conditions, which is consistent with the primary biological roles of the enzymes. CONCLUSIONS: These results demonstrate a functional relationship between aminoglycoside and protein kinases, expanding on our previous observations of similarities in protein structure, enzyme mechanism and sensitivity to inhibitors, and suggest an evolutionary link between APHs and EPKs.     

Stimulation of cell elongation in teleost rod photoreceptors by distinct protein kinase inhibitors

The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)-3beta (a proline-directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3beta and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.     

Constitutive activation of Mek1 by mutation of serine phosphorylation sites

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.     

Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1

Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.     

Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq

The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.     

Phosphorylation of vitronectin by casein kinase II. Identification of the sites and their promotion of cell adhesion and spreading

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain approximately 3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a Km of 0.5-2 microM (lower than the Vn concentration in blood, 3-6 microM), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the alphav beta3 Vn receptors.