The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.     

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.     

Properties of p19, a novel cAMP-dependent protein kinase substrate protein purified from bovine brain

We report the purification from bovine brain and describe some of the properties of a 19-kDa protein, p19, which we have previously shown to undergo hormone-dependent, cAMP-mediated phosphorylation in several peptide hormone-producing tumor cells. The procedure for purifying p19 to apparent homogeneity utilized ammonium sulfate fractionation, sequential chromatography on DEAE-cellulose and phenyl-Sepharose, followed by fast protein liquid chromatography using a Mono Q and, finally, a C8 reverse-phase column. The yield was 0.3-0.5 mg of p19/kg of brain. The molecular weight (Mr = 19,000) and frictional ratio (f/f0 = 1.87) of p19, which were derived from its Stokes radius (33 A) and sedimentation constant (s20,w = 1.4), suggest that the native form of p19 is an asymmetrically shaped monomer. We provide evidence to suggest that p19 is isolated as a mixture of molecular forms consisting of an unphosphorylated form and of three phosphoforms indicative of multisite phosphorylation. These forms cosedimented on sucrose density gradients and coeluted on gel filtration, hydrophobic chromatography, and reverse-phase fast protein liquid chromatography. They were resolved from each other by anion-exchange chromatography. The unphosphorylated form (pI 6.2) was phosphorylated by catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.5 mol of P/mol of p19, thereby giving rise to the three phosphoforms (pI 5.8, pI 5.6, and pI 5.2, respectively). We conclude that p19 is a novel cAMP-dependent protein kinase substrate protein that is present in brain and in peptide hormone-producing tumor cells. Its function remains to be identified.     

Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement

To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dependent endothelial cell barrier regulation, we investigated the impact of the cAMP/PKA inhibitors Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery and bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stress fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotype evolves in an MLC-independent fashion. We next examined the role of extracellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Raf-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf-1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI-induced ERK activation, caldesmon phosphorylation, and stress fiber formation. In summary, our data elucidate the involvement of the p42/44 ERK pathway in cytoskeletal rearrangement evoked by reductions in PKA activity and suggest the involvement of significant cross talk between cAMP- and ERK-dependent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.     

cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.     

Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.     

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.     

CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.     

Bruton's tyrosine kinase associates with the actin-based cytoskeleton in activated platelets

Bruton's tyrosine kinase (Btk) plays a crucial role in the maturation and differentiation of B-lymphocytes and immunoglobulin synthesis. Recently Btk has been described to be present in significant amount in human platelets. To investigate the regulation of this kinase in the platelets we studied its subcellular redistribution in the resting and activated cells. In the resting platelets Btk was almost absent from the actin-based cytoskeleton. Upon challenge of the platelet thrombin receptor upto 30% of total Btk appeared in the cytoskeleton and the protein underwent phosphorylation on tyrosine. Translocation of Btk to the cytoskeleton but not aggregation was prevented by cytochalasin B, which inhibits actin polymerization. Wortmannin and genistein (inhibitors of phosphoinositide 3-kinase and protein tyrosine kinase, respectively) decreased while phenylarsine oxide (a tyrosine phosphatase inhibitor) increased the cytoskeletal content of Btk. The association of Btk with the cytoskeleton was regulated by integrin alpha(IIb)beta(3) and partly reversible. Taken together, these data suggest that Btk might be a component of a signaling complex containing specific cytoskeletal proteins in the activated platelets.     

Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells

The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility.     

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.     

Partial purification and characterization of thrombolamban, a 22,000 dalton cAMP-dependent protein kinase substrate in platelets

In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.     

cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.     

Use of a double-headed peptide substrate to study the specificity of cAMP-dependent protein kinase and posphorylase kinase.

A peptide containing 2 seryl residues, (1)Leu(2)Ser(3)Tyr(4)Arg(5)Aly(6)Tyr(7)Ser(8)Leu, was chemically synthesized and used as a substrate for phosphorylase kinase and cyclic AMP-dependent protein kinase. The sequence, TryArgGlyTyr, makes up a beta turn in the native protein. Phosphorylase kinase was found to phosphorylate specifically seryl residue2 and protein kinase seryl residue7. Km and Vmax values were obtained and compared with natural substrates. The differences in the specificity of the two enzymes might be explained by a different requirement for organized structure. As a working hypothesis, it is suggested the results could be explained if the two enzymes interacted with seryl residues at different sides of a beta turn.     

cAMP-dependent protein kinase I, a unique allosteric enzyme

cAMP-dependent protein kinases are known to be activated by dissociation. There are two types of these enzymes in the mammalian cytosol with similar catalytic subunits but regulatory subunits. With enzymes of type I, ATP counteracts the activation by cAMP. Recent studies of the binding sites of these enzymes for these ligands are reviewed.     

Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.     

Phosphorylation of smg p21B/rap1B p21 by cyclic GMP-dependent protein kinase.

smg p21B/rap1B p21, a member of ras p21-like small GTP-binding protein superfamily, has been shown to be phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP-dependent protein kinase (protein kinase G) in a cell-free system. The same serine residue (Ser179) in the C-terminal region was phosphorylated by both protein kinases G and A. The Km and Vmax values of smg p21B for protein kinase G were 5 x 10(-7) M and 4 x 10(-9) mol/min/mg, and those values for protein kinase A were 1 x 10(-7) M and 3 x 10(-8) mol/min/mg.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.