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This Article Full Text Full Text (PDF) [Supplemental Figures] All Versions of this Article: 78/1/184    most recent biolreprod.107.062943v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fiedler, S. E Articles by Carr, D. W Search for Related Content PubMed PubMed Citation Articles by Fiedler, S. E Articles by Carr, D. W Agricola Articles by Fiedler, S. E Articles by Carr, D. W

Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa¹

Department of Medicine, Oregon Health & Sciences University and Veterans Affairs Medical Center, Portland, Oregon 97239

ABSTRACT

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.

AKAP, calcium, gamete biology, kinases, PKA, RHO, signal transduction, sperm

¹Supported by the Department of Veterans Affairs Biomedical Laboratory Research and Development Service, National Institutes of Health grant HD36408 to D.W.C., the Proteomics Shared Resource, which is funded by the Oregon Opportunity, and by National Institutes of Health center grants 5P30CA069533 and 5P30EY010572.

Correspondence: ²Correspondance: Daniel W. Carr, Veterans Affairs Medical Center, Mail Code R&D8, 3710 SW US Veterans Hospital Rd., Portland, OR 97239. FAX: 503 721 1082; e-mail: carrd{at}ohsu.edu