Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.     

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.     

Enzymatic phosphorylation of food proteins by purified and recombinant protein kinase CK2

Protein kinase CK2 formerly called casein kinase II is a protein kinase able to phosphorylate more than 100 proteic substrates. We have purified protein kinase CK2 from the yeast Y. lipolytica to phosphorylate milk and plant reserve proteins to a significant extent. In the case of plant reserve proteins, which are polymeric substrates, not all subunits are substrate for protein kinase CK2, even if non phosphorylated subunits contain significant potent phosphorylations sites. Best substrates were soy beta-conglycinin (0.72 P/mol) and dephosphorylated caseins (0.5 P/mol). We have studied some functional properties of phosphorylated caseins. Solubility was improved for all pH values but pI. Sensitivity to calcium has also been assessed, and it is slightly improved upon phosphorylation. We have cloned the catalytic subunit of protein kinase CK2 from yeast Y. lipolytica. The recombinant catalytic subunit expressed in E. coli was active and displayed kinetic properties similar to those of the purified enzyme. The recombinant catalytic subunit was able to phosphorylate plant reserve proteins and milk proteins to a significant extent. Best substrates were soy beta-conglycinin (1.0 P/mol), and glycinin (0.59 P/mol).     

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.     

A dopamine/D1 receptor/protein kinase A/dopamine- and cAMP-regulated phosphoprotein (Mr 32 kDa)/protein phosphatase-1 pathway regulates dephosphorylation of the NMDA receptor

We have investigated the mechanism by which activation of dopamine (DA) receptors regulates the glutamate sensitivity of medium spiny neurons of the nucleus accumbens. Our results demonstrate that DA regulates the phosphorylation state of the NR1 subunit of NMDA-type glutamate receptors. The effect of DA was mimicked by SKF82526, a D1-type DA receptor agonist, and by forskolin, an activator of cAMP-dependent protein kinase (PKA), and was blocked by H-89, a PKA inhibitor. These data indicate that DA increases NR1 phosphorylation through a PKA-dependent pathway. DA-induced phosphorylation of NR1 was blocked in mice bearing a targeted deletion of the gene for dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32), a phosphoprotein that is a potent and selective inhibitor of protein phosphatase-1, indicating that the effect of PKA is mediated, in part, by regulation of the DARPP-32/protein phosphatase-1 cascade. In support of this interpretation, NR1 phosphorylation was increased by calyculin A, a protein phosphatase-1/2A inhibitor. A model is proposed in which the ability of DA to regulate NMDA receptor sensitivity is attributable to a synergistic action involving increased phosphorylation and decreased dephosphorylation of the NR1 subunit of the NMDA receptor.     

Inhibition of translation by mRNA encoding NIPP-1, a nuclear inhibitor of protein phosphatase-1

Transient transfection of COS-1 cells with an expression vector for NIPP-1, a nuclear subunit of protein phosphatase-1, did not result in an overexpression of NIPP-1 protein, although the levels of mRNA encoding NIPP-1 increased dramatically. Moreover, high concentrations of NIPP-1 mRNA inhibited the translation in reticulocyte lysates of various unrelated mRNAs. This inhibition of translation was caused by the NIPP-1 messenger and not by the translation product, since mutation of the start codon abolished NIPP-1 protein production, but had no influence on the translational inhibition. Analysis of deletion mutants showed that the inhibition was mediated by a 0.5-kb fragment in the 5'-end of the NIPP-1 mRNA. This region, when inserted in the 5'-untranslated region of the beta-galactosidase messenger, inhibited the translation of beta-galactosidase mRNA in COS-1 cells. A predicted highly stable secondary structure deltaG = -239.5 kJ/mol) is present between residues 300 and 500 of NIPP-1 mRNA. The possible importance of this structure in the translational inhibition is discussed.     

Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways

BACKGROUND: The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase. MATERIALS AND METHODS: The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase. RESULTS: The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. CONCLUSIONS: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.     

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs

The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.     

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.     

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser70) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). In addition, an intact Ser70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A.     

Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.     

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.     

Serine 16 of stathmin as a cytosolic target for Ca2+/calmodulin-dependent kinase II after CD2 triggering of human T lymphocytes

We investigated specific signaling events initiated after T cell triggering through the costimulatory surface receptors CD2 and CD28 as compared with activation via the Ag receptor (TCR/CD3). We therefore followed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphoprotein proposed as a general relay integrating diverse intracellular signaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmodulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. We addressed the specific protein kinase systems involved in the CD2 pathway of T cell activation through the analysis of stathmin phosphorylation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. The partial redundancy of the three T cell activation pathways was evidenced by the phosphorylation of Ser25 and Ser38, substrates of MAP kinases and of the cdk family kinase(s), respectively. Conversely, the phosphorylation of Ser16 of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser16 of recombinant human stathmin was phosphorylated also by purified CaM kinase II, and in vivo, CaM kinase II activity was indeed stimulated in CD2-triggered Jurkat cells. Altogether, our results favor an association of CaM kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser16.     

Characterization of a proline-directed casein kinase from bovine brain

A messenger-independent ser/thr casein kinase, p45 casein kinase (p45 CK), was purified to homogeneity from bovine brain. The enzyme is specific for ATP with a Km value of 3.50 microM, one of the lowest values identified for protein kinases, p45 casein kinase is active over broad NaCl concentrations from 30 to 300 mM. The enzyme activity is inhibited by polylysine, spermine, transition metal ions, ADP, and AMP. The kinase completely lost its activity in the presence of 1 mM p-chloromercuric benzoate in a reaction that is reversed by 1 mM dithiothreitol. The enzyme prefers serine over threonine in its substrate bradykinin, the Vmax/Km ratio for the serine peptide (RPPGFSPFR) being 7.5-fold higher than for the threonine analog (RPPGFTPFR). Assays, performed by utilizing synthetic peptides, suggest that p45 CK prefers serine/threonine residues with a proline residue immediately carboxy-terminal to the site of phosphorylation. Distinction between p45 CK and other protein kinases found to contain a proline residue within their substrate recognition sites can be made based on phosphorylation site specificity and chromatographic and biochemical behavior. It is concluded that p45 CK is a proline-directed protein kinase recognizing the sequence X-Ser/Thr-Pro-X or Ser/Thr-Pro-X.     

Detection of designer drugs in human hair by ion mobility spectrometry (IMS)

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP-dependent kinase creates the more acidic species.     

G-protein beta gamma subunits mediate specific phosphorylation of the protein-tyrosine phosphatase SH-PTP1 induced by lysophosphatidic acid

SH-PTP1 is a protein-tyrosine phosphatase preferentially expressed in hematopoietic cells and bearing two SH2 (src homology-2) domains. In the human megakaryocytic cell line Dami, lysophosphatidic acid (LPA) promoted a rapid increase in SH-PTP1 phosphorylation on both serine and tyrosine residues. Only tyrosine phosphorylation was significantly inhibited by pertussis toxin and by the protein kinase C inhibitor GF109203X. Moreover, SH-PTP1 was phosphorylated upon challenge with other agonists acting via G-protein-coupled receptors such as alpha-thrombin, epinephrine, and ADP, whereas the closely related protein-tyrosine phosphatase SH-PTP2 failed to share such a regulation in Dami cells. We developed an in vitro assay that reproduced LPA-dependent phosphorylation of SH-PTP1 in a cell-free system. The fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1-(495-689) or the transducin subunit Galphat-GDP, which act as specific antagonists of Gbetagamma, inhibited SH-PTP1 phosphorylation. Moreover, purified transducin Gbetagamma subunits mimicked the effect of LPA. Finally, stable expression of beta-adrenergic receptor kinase 1-(495-689) in Dami cells resulted in the inhibition of SH-PTP1 as a specific target of protein kinases linked to G-protein-coupled receptors via Gbetagamma subunits.     

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

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.