Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets

Larry D. Bozulic; Mohammad T. Malik; David W. Powell; Adrian Nanez; Andrew J. Link; Kenneth S. Ramos; William L. Dean

doi:10.1160/TH06-08-0438

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(04): 587-597
DOI: 10.1160/TH06-08-0438

Platelets and Blood Cells

Schattauer GmbH

Plasma membrane Ca²⁺-ATPase associates with CLP36, α-actinin and actin in human platelets

Authors

Larry D. Bozulic

¹Department of Biochemistry and Molecular Biology
Mohammad T. Malik

¹Department of Biochemistry and Molecular Biology
David W. Powell

¹Department of Biochemistry and Molecular Biology

²Department of Medicine, University of Louisville, School of Medicine, Louisville, Kentucky, USA
Adrian Nanez

¹Department of Biochemistry and Molecular Biology
Andrew J. Link

³Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
Kenneth S. Ramos

¹Department of Biochemistry and Molecular Biology
William L. Dean

¹Department of Biochemistry and Molecular Biology

Abstract

Summary

The plasma membrane Ca²⁺-ATPase (PMCA) plays an essentialrole in maintaining low cytosolic Ca²⁺ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts viaits C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the lastten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pumpto the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitationassays coupled with liquid chromatography/tandemmass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing proteinassociated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extractusing a fusion protein containing the C-terminal PDZ domainbinding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized plateletsdemonstrated the existence of a 1,000-kDa complex containingPMCA and CLP36, and in addition, α -actinin and actin. Immunoflourescencemicroscopy confirmed the co-localization ofPMCA with CLP36 in resting and activated platelets. Taken togetherthese results suggest that PMCA is localized in non-filamentousactin complexes in resting platelets by means of PDZdomain interactions and then associates with the actin cytoskeletonduring cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine andtyrosine phosphorylation events previously described in humanplatelets, PMCA function may be regulated by interactions withanchoring and cytoskeletal proteins.

Keywords

Platelets - plasma membrane Ca²⁺-ATPase - calcium signaling - filopodia

Full Text

References

References
1 Carafoli E. The ambivalent nature of the calcium signal. J Endocrinol Invest 2004; 27: 134-139.
2 Rink TJ, Sage SO. Calcium signaling in human platelets. Annu Rev Physiol 1990; 52: 431-439.
3 Butenas S, Cawthern KM, Veer C. et al. Antiplatelet agents in tissue factor-induced blood coagulation. Blood 2001; 97: 2314-2322.
4 Rosado JA, Sage SO. Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets. J Biol Chem 2000; 275: 19529-19535.
5 Lehotsky J, Kaplan P, Murin R. et al. The role of plasma membrane Ca2+ pumps (PMCAs) in pathologies of mammalian cells. Front Biosci 2002; 7: d53-d84.
6 Strehler EE, Zacharias DA. (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiological Reviews 2001; 81: 21-50.
7 Dean WL, Chen D, Brandt PC. et al. Regulation of platelet plasma membrane Ca2+-ATPase by cAMP-dependent and tyrosine phosphorylation. J Biol Chem 1997; 272: 15113-15119.
8 Paszty K, Kovacs T, Lacabaratz-Porret C. et al. Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets. Cell Calcium 1998; 24: 129-135.
9 Carafoli E. Biogenesis: plasma membrane calcium ATPase: 15 years of work on the purified enzyme. FASEB J 1994; 8: 993-1002.
10 Monteith GR, Roufogalis BD. The plasma membrane calcium pump--a physiological perspective on its regulation. Cell Calcium 1995; 18: 459-470.
11 Schwab BL, Guerini D, Didszun C. et al. Cleavage of plasma membrane calcium pumps by Caspases: a link between apoptosis and necrosis. Cell Death Differ 2002; 9: 818-831.
12 Filomatori CV, Rega AF. On the mechanism of activation of the plasma membrane Ca 2+-ATPase by ATP and acidic phospholipids. J Biol Chem 2003; 278: 22265-22271.
13 Valverde RH, Tortelote GG, Lemos T. et al. Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca 2+-ATPase by calmodulin and protein kinase A. J Biol Chem 2005; 280: 30611-30608.
14 Blankenship KA, Dawson CB, Aronoff GR. et al. Tyrosine phosphorylation of human platelet plasma membrane Ca 2+-ATPase. Hypertension 2000; 35: 103-107.
15 Wan TC, Zabe M, Dean WL. Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets. Thromb Haemost 2003; 89: 122-131.
16 Fox JE. The platelet cytoskeleton. Thromb Haemost 1993; 70: 884-893.
17 Fox JE, Shattil SJ, Kinlough-Rathbone RL. et al. The platelet cytoskeleton stabilizes the interaction between α IIbβ 3 and its ligand and induces selective movements of ligand-occupied integrin. J Biol Chem 1996; 271: 7004-7011.
18 Fox JEB, Boyles JK, Reynolds CC. et al. Actin filament content and organization in unstimulated platelets. J Cell Bio 1984; 98: 1985-1991.
19 Eigenthaler M, Shattil SJ. Integrin signaling and the platelet cytoskeleton. Current Topics Membranes 1996; 43: 265-291.
20 Garner CC, Nash J, Huganir RL. PDZ domains in synapse assembly and signaling. Trends Cell Biol 2000; 10: 274-280.
21 Kim E, DeMarco SJ, Marfatia SM. et al. Plasma membrane Ca 2+-ATPase isoform 4b binds to membrane- associated guanylate kinase (MAGUK) proteins via their PDZ (PSD-95/Dlg/ZO-1) domains. J Biol Chem 1998; 273: 1591-1595.
22 DeMarco SJ, Strehler EE. Plasma membrane Ca2+-ATPase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane- associated guanylate kinase family of PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. J Biol Chem 2001; 276: 21594-21600.
23 Zabe M, Dean WL. Plasma membrane Ca 2+-ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain. J Biol Chem 2001; 276: 14704-14709.
24 Dean WL, Whiteheart SW. Plasma membrane Ca2+-ATPase (PMCA) translocates to filopodia during platelet activation. Thromb Haemost 2004; 91: 325-333.
25 Bozulic LD, Brown CS, Powell DW. et al. Regulation of plasma membrane Ca2+-ATPase in human platelets. FASEB J 2005; 20: 610-5. Abstract
26 Bauer K, Kratzer M, Otte M. et al. Human CLP36, a PDZ-domain and LIM-domain protein, binds to α -actinin- 1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells. Blood 2000; 96: 4236-4245.
27 Schultess J, Danielewski O, Smolenski AP. RAP1-GAP2 is a new GTPase-activating protein of Rap1 expressed in human platelets. Blood 2005; 105: 3185-3192.
28 Schneider C, Newman RA, Sutherland R et al. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem 1982; 257: 10766-10769.
29 Laemmli UK. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970; 227: 680-685.
30 Sanders SL, Jennings J, Canutescu A. et al. Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry. Mol Cell Biol 2002; 22: 4723-4738.
31 Eng JK, McCormack AL, Yates JR. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J Am Soc Mass Spectrom 1994; 5: 976-989.
32 Link AJ, Eng J, Schieltz DM. et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999; 17: 676-682.
33 Powell DW, Weaver CM, Jennings JL et al. Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Mol Cell Biol 2004; 24: 7249-7259.
34 Franzot G, Sjoblom Gautel M. et al The crystal structure of the actin binding domain from α -actinin in its closed conformation: structural insight into phospholipid regulation of α -actinin. J Mol Biol 2005; 348: 151-165.
35 Miehe U, Kadyrov M, Neumaier-Wagner P. et al. Expression of the actin stress fiber-associated protein CLP36 in the human placenta. Histochem Cell Biol 2006. doi: 10.1007/s00418–006–0182–5.
36 Ktaka g M, Lau Y, Cheung KK. et al. Elfin is expressed during early heart development. J Cell Biochem 2001; 83: 463-472.
37 Zheng JQ. Turning of nerve growth cones induced by localized increases in intracellular calcium ions. Nature 2000; 403: 89-93.
38 McAfee KJ, Duncan DT, Assink M. et al. Analyzing proteomes and protein function using graphical comparative analysis of tandem mass spectrometry results. Mol Cell Proteomics 2006; 5: 1497-1513.
39 Powell DW, Weaver CM, Jennings JL. et al. Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Mol Cell Biol 2004; 24: 7249-7259.