Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1
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BIOLOGY OF REPRODUCTION 78, 184–192 (2008) Published online before print 10 October 2007. DOI 10.1095/biolreprod.107.062943 Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1 Sarah E. Fiedler, Malini Bajpai, and Daniel W. Carr2 Department of Medicine, Oregon Health & Sciences University and Veterans Affairs Medical Center, Portland, Oregon 97239 ABSTRACT Guanine nucleotide exchange factors (GEFs) catalyze the GDP for GTP exchange [2]. Activation is negatively regulated by In somatic cells, RHOA mediates actin dynamics through a both guanine nucleotide dissociation inhibitors (RHO GDIs) GNA13-mediated signaling cascade involving RHO kinase and GTPase-activating proteins (GAPs) [1, 2]. Endogenous (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be RHO can be inactivated via C3 exoenzyme ADP-ribosylation, negatively regulated by protein kinase A (PRKA), and it and studies have demonstrated RHO involvement in actin-based interacts with members of the A-kinase anchoring (AKAP) cytoskeletal response to extracellular signals, including lyso- family via intermediary proteins. In spermatozoa, actin poly- merization precedes the acrosome reaction, which is necessary phosphatidic acid (LPA) [2–4]. LPA is known to signal through for normal fertility. The present study was undertaken to G-protein-coupled receptors (GPCRs) [4, 5]; specifically, LPA- determine whether the GNA13-mediated RHOA signaling activated GNA13 (formerly Ga13) promotes RHO activation pathway may be involved in acrosome reaction in bovine through GEFs [4, 6]. Activated RHO-GTP then signals RHO caudal sperm, and whether AKAPs may be involved in its kinase (ROCK), resulting in the phosphorylation and activation targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and of LIM-kinase (LIMK), which in turn phosphorylates and cofilin were all detected by Western blot in bovine caudal inactivates cofilin, an actin depolymerizer, the end result being sperm. Overlay, immunoprecipitation, and subsequent mass actin polymerization [7–9] (See Supplemental Fig. 1 for spectrometry analysis identified several RHOA-interacting diagram available online at www.biolreprod.org). proteins, including proacrosin, angiotensin-converting enzyme, A-kinase anchoring proteins (AKAPs) are defined by their tubulin, aldolase C, and AKAP4. Using overlay and pulldown ability to bind one or more of the regulatory subunits techniques, we demonstrate that phosphorylation of AKAP3 (PRKAR1A, PRKAR2A, PRKAR1B, and PRKAR2B, former- increases its interaction with the RHOA-interacting proteins ly RIa, RIIa,RIb, and RIIb) of cAMP-dependent protein PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) kinase A (PRKA); these subunits interact with an amphipathic and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). helix on the AKAP. AKAPs target the action of PRKA by Varying calcium concentrations in pulldown assays did not acting as scaffolding proteins, spatially restricting function by significantly alter binding to R2D2 proteins. These data suggest simultaneously binding several related signal transduction that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK- enzymes [10–13]. Activation of PRKA usually results from cofilin pathway is present in bovine spermatozoa, that RHOA the binding of cAMP to the R subunits of PRKA, which interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be promotes dissociation and activation of the catalytic subunits, targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and leading to a wide variety of cellular responses. However, ROPN1 is regulated by phosphorylation in vitro. AKAP3 can simultaneously bind PRKA and activated GNA13, causing the dissociation of the catalytic subunits of PRKA, thus AKAP, calcium, gamete biology, kinases, PKA, RHO, activating PRKA in a cAMP-independent manner [14]. PRKA signal transduction, sperm activation has been shown to negatively regulate RHOA signaling in two ways: PRKA directly phosphorylates RHOA, INTRODUCTION leading to increased interaction with RHO-GDI and transloca- The RHO signaling pathway is involved in stimulating actin tion from the membrane to the cytosol [15–17], and PRKA polymerization, a process that regulates many cellular functions, phosphorylates GNA13, substantially reducing RHO activation including cell division, motility, and polarity [1]. The RHO (an effect that was blocked by the introduction of a GNA13 family of signaling molecules is a group of small GTP-binding mutant incapable of PRKA-mediated phosphorylation) [18]. proteins within the Ras-related small GTPase superfamily. RHO Ht31, an anchoring inhibitor peptide (AIP) that competitively GTPases are present in all eukaryotic cells, where they alternate disrupts PRKA binding to AKAPs, blocks PRKA phosphor- between inactive GDP-bound and active GTP-bound states. ylation of RHO and prevents PRKA-induced inhibition of RHOA in human SGC-7901 cells [19]. 1Supported by the Department of Veterans Affairs Biomedical A yeast two-hybrid screen using AKAP3 as bait identified Laboratory Research and Development Service, National Institutes of two other proteins that interact with the amphipathic helix in a Health grant HD36408 to D.W.C., the Proteomics Shared Resource, manner similar to PRKAR2: AKAP-associated sperm protein which is funded by the Oregon Opportunity, and by National Institutes (ROPN1L, formerly ASP, a novel protein) and ropporin of Health center grants 5P30CA069533 and 5P30EY010572. 2 (ROPN1), a protein involved in the RHO pathway via interaction Correspondance: Daniel W. Carr, Veterans Affairs Medical Center, Mail with rhophilin [20, 21]. Due to the sequence similarity of Code R&D8, 3710 SW US Veterans Hospital Rd., Portland, OR 97239. FAX: 503 721 1082; e-mail: [email protected] ROPN1L and ROPN1 to the N-terminal dimerization and docking domain of RII, these proteins have been named R2D2 proteins [22]. Sequence analysis identified two other proteins Received: 16 May 2007. that contain this R2D2 domain: sperm protein 17 (SPA17) and First decision: 8 June 2007. Accepted: 26 September 2007. calcium-binding tyrosine phosphorylation-regulated protein Ó 2008 by the Society for the Study of Reproduction, Inc. (CABYR, formerly FSII) [23, 24]. All four of these proteins ISSN: 0006-3363. http://www.biolreprod.org were originally identified as sperm-specific proteins [22]. 184 RHOA-INTERACTING PROTEINS IN BOVINE SPERMATOZOA 185 In bovine spermatozoa, redistribution of actin-regulating Chelating HP columns were from Amersham Biosciences (Uppsala, Sweden). proteins and increased actin polymerization takes place during S-protein agarose, S-protein horseradish peroxidase conjugate, and BL21(dE3)- capacitation and is suggested to have a role in the acrosome pLysS competent cells were from Novagen (Madison, WI). GTPcS was from Cytoskeleton (Denver, CO). Immobilon-P PVDF membrane was from Millipore reaction (AR) [25–27]. Brener et al. [26] have shown that C3 Corp. (Bedford, MA). Phospho-(Ser/Thr) PRKA substrate antibody (monoclo- exoenzyme inhibition of RHO blocks capacitation-induced nal), cofilin, and LIMK2 antibodies were from Cell Signaling Technologies actin polymerization and subsequent acrosome reaction. The (Beverly, MA). Production of a-AKAP3 antibody (rabbit, polyclonal) was authors suggest that cofilin inactivation via RHO signaling may described previously in Vijayaraghavan et al. [11]. Rabbit IgG and a-ROCK2 be involved in this process. However, GNA13, the G protein (monoclonal) antibody were from BD Pharmingen (San Diego, CA). Goat a- that mediates cofilin signaling through RHOA, has not been rabbit and goat a-mouse horseradish peroxidase-conjugated secondary antibod- ies, polyclonal a-RHOA(119), polyclonal a-ROCK2 (H-85), and polyclonal a- previously demonstrated in sperm. RHOA and ROCK1 are GNA13 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). known to be present in sperm, and have been localized to both Western Lightning chemiluminescence and 32P-ATP were from Perkin Elmer the head and the tail of bovine spermatozoa [21, 28]. LIMK2 (Boston, MA). Modified trypsin for mass spectrometry sample preparation was and cofilin are present in testes, but they have not yet been from Promega. Sonication was performed using the Sonic Dismembrator 60 specifically identified in spermatozoa [29, 30]. It has been from Fisher Scientific (Pittsburgh, PA). PRKA (cat) was kindly supplied by the suggested that cofilin and LIMK signaling requires tight spatial Susan Taylor lab. Bovine testes came from Carlton Farms, (Carlton, OR), and ejaculated spermatozoa (cryopreserved) were from Select Sires Inc. (Plain City, regulation [1, 8]. As scaffolding molecules known to associate OH). Investigations involving animals were conducted in accordance with with RHOA pathway members, AKAPs are potential candi- ‘‘Guide for Care and Use of Laboratory Animals.’’ dates for this targeting [14, 20, 21, 31–35]. To date, six AKAPs have been identified in spermatozoa: AKAP1, AKAP3, Western Blotting AKAP4, AKAP11, MAP2, and WAVE1 [13, 36–38]. Bovine caudal spermatozoa were obtained as described previously [12]. For Although much work has been conducted on the regulation RHOA and ROCK2 Western blots, 4.0 3 108 spermatozoa were lysed in 250 ll of proteins associated with AKAPs (i.e., PRKA phosphoryla- buffer 6 (5 mM Tris, pH 7.6, 0.1% Triton X-100, 250 mM sucrose, 1 mM tion of RHOA has an inhibitory effect [15–18]), studies on sodium vanadate, 1 mM sodium fluoride, 5 ng/ml leupeptin, 1 mM