PKA phosphorylation of Src mediates cAMP's inhibition of cell growth via Rap1.

In fibroblast cells, cAMP antagonizes growth factor activation of ERKs and cell growth via PKA and the small G protein Rap1. We demonstrate here that PKA's activation of Rap1 was mediated by the Rap1 guanine nucleotide exchange factor C3G, the adaptor Crk-L, the scaffold protein Cbl, and the tyrosine kinase Src. Src was required for cAMP activation of Rap1 and the inhibition of ERKs and cell growth. PKA activated Src both in vitro and in vivo by phosphorylating Src on serine 17 within its amino terminus. This phosphorylation was required for cAMP's activation of Src and Rap1, as well as cAMP's inhibition of ERKs and cell proliferation. This study identifies an antiproliferative role for Src in the physiological regulation of cell growth by cAMP.     

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.     

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.     

血管抑制素功能结构域K1与PKA底物磷酸化基序的融合表达及磷酸化标记

通过 RT- PCR,从人肝组织中扩增出血管形成抑制素 ( angiostatin) c DNA的 K1片段 ,经DNA序列分析证实其正确性 ;将 K1与 GST融合并带上 1 7个氨基酸的 PKA底物磷酸化基序 ,IPTG诱导表达 ,以还原型谷胱甘肽偶联的琼脂糖凝胶亲合层析直接从细菌裂解上清中纯化融合蛋白 ;以 PKA催化单位将 3 2 P通过磷酸化作用标记至纯化的蛋白 ,再用凝血酶切去 GST,进行SDS- PAGE.放射自显影结果显示 ,GSTag- K1和 Tag- K1分别在 40 k D和 1 7k D处有信号强而特异的显影条带 ,表明带有磷酸化序列的蛋白能够被 PKA特异地磷酸化标记     

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.     

Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C

The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles. These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.     

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.     

cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.     

Cyclic adenosine 5'-monophosphate-stimulated neurotensin secretion is mediated through Rap1 downstream of both Epac and protein kinase A signaling pathways

Neurotensin (NT), a gut peptide, plays important roles in gastrointestinal secretion, inflammation, and growth of normal and neoplastic tissues. cAMP regulates the secretion of hormones via its effector proteins protein kinase A (PKA) or Epac (exchange protein directly activated by cAMP). The small GTPase Rap1 can be activated by both PKA and Epac; however, the role of Rap1 in hormone secretion is unknown. Here, using the BON human endocrine cell line, we found that forskolin (FSK)-stimulated NT secretion was reduced by inhibition of Rap1 expression and activity. FSK-stimulated NT secretion was enhanced by overexpression of either wild-type or constitutively active Rap1. Epac activators and wild-type Epac enhanced NT release and Rap1 activity. In contrast, overexpression of a cAMP binding mutant, EpacR279E, decreased NT release and Rap1 activity. PKA activation increased NT release and Rap1 activity. FSK-stimulated NT release was reduced by PKA inhibition and the dominant negative Rap1N17. NT secretion, stimulated by Epac activation, was reduced by PKA inhibition; NT release, stimulated by PKA activation, was enhanced by wild-type Epac but reduced by the mutant EpacR279E. Finally, prostaglandin E2 (PGE2), a physiological agent that increases cAMP, stimulated NT secretion via cAMP/PKA/Rap1. Importantly, we demonstrate that PKA and Epac mediate the cAMP-induced NT secretion synergistically by converging at the common downstream target protein Rap1. Moreover, PGE2, a potent mediator of inflammation and associated with colorectal carcinogenesis, stimulates NT release suggesting a possible link between PGE2 and NT on intestinal inflammatory disorders and colorectal cancers.     

In vitro phosphorylation by cAMP-dependent protein kinase up-regulates recombinant Saccharomyces cerevisiae mannosylphosphodolichol synthase

DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.

We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.     

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

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

Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation

cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.     

Molecular approaches to examine the phosphorylation state of the C type natriuretic peptide receptor

The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST‐NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine‐5'‐triphosphate (ATP). Western blot analysis using a monoclonal phospho‐Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross‐react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation. J. Cell. Biochem. 110: 985–994, 2010. © 2010 Wiley‐Liss, Inc.     

A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

In 32Pi-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. It was purified from bovine neutrophil cytosol by a series of chromatographic steps, including ion exchange on DE-52 cellulose and Mono Q, and filtration on Bio-Gel P60 in the presence of mercaptoethanol and urea. The apparent molecular mass of the purified protein, assessed by SDS-PAGE and mercaptoethanol by reference to protein markers, ranged between 20 and 23 kDa, depending on the percentage of polyacrylamide and conditions of migration. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Some properties of the 23-kDa protein, including its amino acid composition, were determined. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of four discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [gamma-32P]ATP in the presence of bovine neutrophil PKC supplemented with Ca2+, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. The apparent KM of ATP was 9 microM. The 23-kDa protein was also phosphorylated by PKM, the catalytic fragment of PKC obtained after removal of the regulatory domain, but not by cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.