Abstract
Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.
Highlights
From the ‡Division of Cardiology, University of Manchester, Manchester M13 9PT, United Kingdom and the ¶Department of Cell and Cancer Biology, NCI, National Institutes of Health, Rockville, Maryland 20850-3300
As a first step to address this hypothesis, we decided to identify proteins that would bind to the region 652– 840 of the second intracellular loop of human PMCA4b
In this work we have focused on the characterization of the interaction between PMCA4b and RASSF1
Summary
Vol 279, No 30, Issue of July 23, pp. 31318 –31328, 2004 Printed in U.S.A. Novel Functional Interaction between the Plasma Membrane Ca2؉ Pump 4b and the Proapoptotic Tumor Suppressor. Our group and others have identified other molecular interactions involving the COOH-terminal region and PDZ domaincontaining proteins These proteins include members of the membrane-associated guanylate kinase (MAGUK) family [15, 16], cytoskeletal proteins [17], Naϩ/Hϩ exchanger regulatory factor 2 [18], nitric-oxide synthase I (nNOS) [19], calcium/ calmodulin-dependent serine protein kinase (CASK) [20], and a novel protein named PISP (PMCA-interacting single-PDZ protein) [21]. The existence of other intracellular domains in the PMCA polypeptide chain, and the previous reports of a functional interaction between PMCA and calmodulin (a protein without PDZ domains), prompted us to investigate whether other regions are implicated in interactions with different partner proteins To test this hypothesis, we performed a two-hybrid screen of a human fetal heart cDNA library. It suggests a role for PMCA as an organizer of macromolecular protein complexes, and a regulator of intracellular signaling pathways in the microenvironment of these multiprotein complexes
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