Distribution and Activation of cAMP- and cGMP-Dependent Protein Kinases in Highly Purified Human Platelet Plasma and Intracellular Membranes

Summary

Previously cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK) have been found predominantly associated with the particulate fraction in human platelets. We now report the distribution and activation of cAMP-PK and cGMP-PK in highly purified fractions of human platelet plasma (PM) and intracellular membranes (IM) prepared using high voltage free flow electrophoresis. Two non-hydrolys-able analogues of cAMP and cGMP namely Sp-5,6-DCl-cBiMPS and 8-p-CPT-cGMP have been used to activate cAMP-PK and cGMP-PK respectively. Addition of either agonist with [γ³²P]ATP stimulated the endogenous activity of cAMP-PK or cGMP-PK in PM but not in IM. With PM Sp-5,6-DCl-cBiMPS stimulated the phosphorylation of protein substrates of Mr 16,22,24,46-50,66,90,160 and 250 kDa. A specific peptide inhibitor of cAMP-PK inhibited the phosphorylation of all of the substrates by Sp-5,6-DCl-cBiMPS. 8-pCPT-cGMP also induced the phosphorylation of a number of substrates particularly 16,22, 46-50, 90 and 250 kDa proteins. Inclusion of the cAMP-PK inhibitor peptide totally blocked the phosphorylation of the 16 and 22 kDa proteins, partially inhibited phosphorylation of 46-50 and 90 kDa proteins and had no effect on the 250 kDa protein indicating the 46-50, 90 and 250 kDa proteins were also cGMP-PK substrates. Western blotting with antibodies to cGMP-PK and the catalytic subunit of cAMP-PK revealed the presence of the kinases to be exclusively associated with PM with no detection in IM.

The presence of cAMP-PK substrates in IM was investigated by exogenous addition of catalytic subunit of cAMP-PK. Phosphoproteins of Mr 16, 22, 27, 30,45, 75,116 and 250 kDa were detected. A range of antibodies to cAMP-PK substrates were used to identify and localise the substrates. These antibodies revealed GPIb and VASP to be exclusively associated with PM fractions. Rap IB was also predominantly associated with PM with a small level detected in IM. Antibodies to the IP3 receptor (18A10 and 4C11) revealed the protein to be predominantly associated with IM. Additionally the antibody 4C11 recognised a 230 kDa protein band in PM that was not seen in IM. From the known specificity of these antibodies the results confirm the presence of a type IIP₃ receptor in IM and a distinct (possible type III) IP₃ receptor with the PM. the 16, 22, 27, 30, 75 and 116 kDa proteins in IM represent nwly detected substartes for camp-pk of presently unknown identity.

• References

• 1 Siess W. Molecular mechanisms of platelet activation. Physiol Rev 1989; 69: 58-178
• 2 Walter U, Eigenthaler M, Geiger J, Reinhard M. Role of cyclic nucleotide-dependent protein kinases and their common substrate VASP in the regulation of human platelets. Adv Exp Med Biol 1993; 344: 237-249
• 3 Rittenhouse-Simmons S. Production of diglyceride from phosphatidy 1-inositol in activated platelets. J Clin Invest 1979; 63: 580-587
• 4 Watson SP, McConnell RT, Lapetina EG. The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J Biol Chem 1984; 259: 13199-13203
• 5 Nakashima Y, Tohmatsu T, Hattori H, Okano Y, Nozawa Y. Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin stimulated human platelets. Biochem Biophys Res Commun 1986; 135: 1099-1104
• 6 Sane DE, Bielawska A, Greenberg CS, Hannum YA. Cyclic GMP analogs inhibit gamma thrombin induced arachidonic acid release. Biochem Biophys Res Commun 1989; 165: 708-714
• 7 Waldmann R, Walter U. Cyclic nucleotide-elevating vasodilators inhibit platelet aggregation at an early step of the activation cascade. Eur J Pharmacol 1989; 159: 317-20
• 8 Siess W, Grunberg B, Luber K. Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition. Adv Exp Med Biol 1993; 344: 299-335
• 9 Waldmann R, Bauer S, Gobel C, Hofmann F, Jakobs KH, Walter U. Demonstration of cGMP dependent protein kinase and cGMP dependent phosphorylation in cell free extracts of platelets. Eur J Biochem 1986; 158: 203-210
• 10 Eigenthaler M, Nolte C, Halbrugge M, Walter U. Concentration and regulation of cyclic nucleotides, cyclic nucleotide-dependent protein kinases and one of their major substrates in human platelets. Eur J Biochem 1992; 205: 471-481
• 11 Siess W, Lapetina EG. Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition. Biochem J 1990; 271: 815-819
• 12 Cox AC, Carrol RC, White JG, Rao GHR. Recycling of platelet phosphorylation and cytoskeletal assembly. J Cell Biol 1984; 98: 8-15
• 13 Halbrugge M, Friedrich C, Eigenthaler M, Schanzenbacher P, Walter U. Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. J Biol Chem 1990 265: 3088-3093
• 14 Fox JEB, Reynolds CC, Johnson MM. Identification of glycoprotein Ib(3 as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase. J Biol Chem 1987; 262 12627-12631
• 15 Siess W, Winegar DA, Lapetina EG. Rap-lb is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun 1990; 170: 944-50
• 16 Fox JEB, Berndt MC. Cyclic AMP-dependent phosphorylation of glycoprotein lb inhibits collagen-induced polymerization of actin in platelets. J Biol Chem 1989; 264: 9520-9526
• 17 Horstrup K, Jablonka B, Honig-Leidl P, Just M, Kochsiek K, Walter U. Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Seri57 ip intact platelets correlates with fibrinogen receptor inhibition. Eur J Biochem 1994; 225: 21-27
• 18 Menashi S, Weintraub H, Crawford N. Characterisation of human platelet surface and intracellular membranes by free flow electrophoresis. J Biol Chem 1981; 256: 4095-4101
• 19 Lagarde M, Guichardant M, Menashi S, Crawford N. The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis. J Biol Chem 1982; 257: 3100-3104
• 20 Hack N, Crawford N. Two dimensional polyacrylamide gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes. Biochem J 1984; 222: 235-246
• 21 Crawford N, Hack N, Authi KS. Isolation and characterisation of platelet membranes prepared by free flow electrophoresis. Meth Enzymol 1992; 215: 5-20
• 22 AuthiZ KS. Ca²⁺ homeostasis and intracellular pools in human platelets. Adv Exp Biol Med 1993; 344: 83-104
• 23 Butt E, Nolte C, Schulz S, Beltman J, Beavo J, Jastorff B, Walter U. Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-_pCPT-_cGMP. Biochem Pharmacol 1992; 43: 2591-2600
• 24 Sandberg M, Butt E, Nolte L, Fischer L, Halbrugge M, Beltman J, Jahnsen T, Genieser HG, Jastorff B, Walter U. Characterization of Sp-5, 6-dichloro-l-΂-D-ribofuranosylbenzimidazole-3’,5’-monophosphorothioate (Sp-5, 6-DC1-cBIMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells. Biochem J 1991; 279: 521-527
• 25 Geiger J, Nolte L, Butt E, Sage SO, Walter U. Role of cGMP and cGMP-depen-dent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. Proc Natl Acad Sci USA 1992; 89: 1031-5
• 26 Menshikov MY, Ivanova K, Schaefer M, Dmmmer C, Gerzer R. Influence of the cGMP analog 8-PCPT-cGMP on agonist induced increases in cytosolic ionized Ca²⁺ and on aggregation of human platelets. Eur J Pharmacol Mol Pharmacol 1993; 245: 281-284
• 27 Lohmann SM, Schwoch G, Reiser G, Port R, Walter U. Dibutyrl cAMP treatment of neuroblastoma-glioma hybrid cells results in selective increase in cAMP-receptor protein (R-I) as measured by monospecific antibodies. EMBO J 1983; 2: 153-159
• 28 Authi KS, Crawford N. Inositol (1, 4, 5) trisphosphate induced release of sequestered calcium from highly purified human platelet intracellular membranes. Biochem J 1985; 230: 247-253
• 29 Cullen PJ, Patel Y, Kakkar VV, Irvine RF, Authi KS. Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membrane of human platelets. Biochem J 1994; 298: 739-42
• 30 Laemmli UK. Cleavage of structural proteins during the assembly of the head by bacteriophage T4. Nature 1970; 227: 680-5
• 31 Cheng H-C, Kemp BE, Pearson RB, Smith AJ, Miscon L, van Patten SM, Walsh DA. A potent synthetic peptide inhibitor of the _cAMP-dependent protein kinase. J Biol Chem 1986; 261: 989-92
• 32 Ohmstede C-A, Farrel FX, Reep BR, Clemetson KJ, Lapetina EG. Rap2B: A ras-related GTP-binding protein from platelets. Proc Natl Acad Sci USA 1990; 87: 6527-6531.
• 33 Wojcikiewicz RJH, Furuichi T, Nakade S, Mikoshiba K, Nahorski SR. Muscarinic receptor activation down-regulates the type I inositol 1,4,5-trisphosphate receptor by accelerating its degradation. J Biol Chem 1994; 269: 7963-7967
• 34 Wojcikiewicz RJH. Type I, II and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem 1995; 270: 11678-11683
• 35 Crawford N, Scrutton MC. Biochemistry of the blood platelet. In: Haemostasis and Thrombosis Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. (eds) Churchill Livingstone; 1994: 89-114
• 36 Ueda M, Oho C, Takisawa H, Ogihawa S. Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the _cAMP-dependent protein kinase in bovine platelets. Eur Biochem 1992; 203: 347-352
• 37 Koga T, Yoshid Y, Cai J-Q, Islam MO, Imai S. Purification and characterizatio of 240 kDa _cGMP-dependent protein kinase substrate of vascular smoot muscle. Close resemblance to Inositol 1,4,5-trisphosphate receptor. J Biol Cher 1994; 269: 11640-11647
• 38 Komalavilas P, Lincoln TM. Phosphorylation of the inositol 1,4,5-triphosphat receptor by cyclic GMP-dependent protein kinase. J Biol Chem 1994; 269: 8701-8707
• 39 Rengasamy A, Feinberg H. Inositol 1,4,5-trisphosphate induced Ca2+ releas from platelet plasma membrane vesicles. Biochem Biophys Res Commun 1988; 150: 1021-1026
• 40 Authi KS. Localisation of the IP3 binding site on platelet membranes isolated b high voltage free flow electrophoresis. FEBS Lett 1992; 298: 173-176
• 41 Fujimoto T, Nakade S, Miyawaki A, Mikoshiba K, Ogawa K. Localization c inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol 1992; 119: 1507-1513
• 42 Winegar DA, MolinayVedia L, Lapetina EG. Isoprenylation of rap2 proteins i platelets and human erythroleukemia cells. J Biol Chem 1991; 266: 4381-6
• 43 Lapetina EG, Farrel FX. Rap IB and platelet function. Adv Exp Biol Med 1993; 344: 49-55
• 44 Corvazier E, Enouf J, Papp B, Gunzburg Jde, Tavitian A, Levy-Toledano S. Evidence for a role of rap 1 protein in the regulation of human platelet Ca²⁺ fluxes. Biochem J 1992; 281: 325-331
• 45 Klinz FJ, Seifert R, Schwauer I, Gausepohl H, Frank R, Schultz G. Generatio of specific antibodies against the rap 1A, rap IB and rap 2 small GTP-bindin proteins. Analysis of rap and ras proteins in membranes from mammalian cells. Eur J Biochem 1992; 207: 207-13
• 46 Butt E, Abel K, Krieiger M, Palm D, Hoppe V, Hoppe J, Walter U. cAMP- an cGMP-dependent protein kinase phosphorylation sites of the focal adhesio vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact huma platelets. J Biol Chem 1994; 269: 14509-14517
• 47 Reinhard M, Halbrugge M, Scheer U, Weigand C, Jockusch BM, Walter U. The 46/50 kDa phosphoprotein VASP purified from human platelets is a novi protein associated with actin filaments and focal contacts. EMBO J 1992; 11: 2063-2070
• 48 Authi KS, Lagarde M, Crawford N. Diacylglycerol lipase activity in huma platelet intracellular and surface membranes. Some kinetic properties and fatl acid specificity. FEBS Lett 1985; 180: 95-101
• 49 Bokkala S, El-Daher SS, Kakkar VV, Wuytack F, Authi KS. Localization an identification of Ca²⁺ ATPases in highly purified human platelet plasma ar intracellular membranes. Evidence that the monoclonal antibody PL/IM 43 recognises the SERCA 3 Ca²⁺ATPase in human platelets. Biochem J 1995; 306: 837-842
• 50 Dean WL, Quinton JM. Distribution of plasma membrane Ca2+ATPase ar inositol 1,4,5-trisphosphate receptor in human platelets. Cell Calcium 1995; 17: 65-70
• 51 Quinton TM, Brown KD, Dean WL. Inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from platelet internal membranes is regulated by differential phosphor lation. Biochemistry 1996; 35: 6865-6871
• 52 Bourguignon LYW, Iiada N, Jin H. The involvement of the cytoskeleton : regulating IP₃ receptor mediated internal Ca²⁺ release in human blood platelets. Cell Biol Intemat 1993; 17: 751-758
• 53 O’Rourke F, Matthews E, Feinstein MB. Purification and characterization of tl human type I Ins(l,4,5)P₃ receptor from platelets and comparison with recepti subtypes in other normal and transformed blood cells. Biochem J 1995; 312: 499-503
• 54 Furuichi T, Kohda K, Miyawaki A, Mikoshiba K. Intracellular channels. Cu Opin Neurobiology 1994; 4: 294-303
• 55 Danoff SK, Ferris CD, Donath C, Fischer G, Munemitsu S, Ullrich A, Snyd SH. Inositol 1,4,5-trisphosphate receptors: Distinct neural and non-neural fom derived by alternative splicing differ in phosphorylation. Proc Natl Acad Sci USA 1991; 88: 2951-2955