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Monash Institute of Medical Research4 and the Australian Research Council (ARC) Centre of Excellence in Biotechnology and Development,5 Monash University, Melbourne, Victoria 3168, Australia
ARC Centre of Excellence in Biotechnology and Development,6 Newcastle University, Newcastle, New South Wales 2308, Australia
ABSTRACT
Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an
60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.
acrosome, CRISP2, male reproductive tract, MAP3K11, sperm, sperm maturation, spermatid, spermatogenesis, testis, TPX1
CRISP2 is a member of the cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis-related (CAP) protein superfamily, and is found within both the sperm acrosome and tail [1–4]. More precisely, CRISP2 is a member of the CRISP subdivision of the CAP superfamily, which is defined by a CAP signature motif common to the entire superfamily, and 10 absolutely conserved cysteine residues in the carboxyl terminus. The cysteines are involved in intramolecular disulphide bonding to form the CRISP domain and, in turn, the CRISP domain is composed of a hinge region and an ion channel regulatory (ICR) region (or cysteine-rich domain) [5, 6].
Within mammals, four CRISPs have been defined: CRISP1 (acidic epididymal glycoprotein [AEG], proteins D and E), CRISP2 (TPX1), CRISP3, and CRISP4. All are strongly expressed within the male reproductive tract, suggesting key roles in male fertility. CRISP1, CRISP2, and CRISP4 are most strongly expressed in the male reproductive tract, where they are produced predominantly in the epididymis, testis, and epididymis, respectively [3, 7]. CRISP3 has a more widespread distribution, including in the salivary gland, pancreas, B cells, and diseased prostate [8–11]. Through the use of cell transfection experiments, CRISP2 has been implicated in adhesion between spermatids and Sertoli cells [12], and CRISP1 and CRISP2 have been implicated in sperm-egg binding [13–16], although the biochemistry underlying these interactions remains to be elucidated.
Analogous to the ion channel activity of CRISPs in the venom of several snakes and lizards [17, 18], the CRISP domain of CRISP2 has recently been shown to be able to regulate Ca2+ influx through ryanodine receptors (RYR) [5]. Unlike reptile CRISPs, however, CRISP2 showed a differential action on RYR subtypes. Specifically, the CRISP2 CRISP domain inhibited RYR2 and activated RYR1 when added to the cytoplasmic domain of the receptor. Furthermore, when added to the luminal domain of the receptor, the CRISP2 CRISP domain activated both RYR1 and RYR2. These data raise the possibility that CRISP2 has a role in the regulation of the Ca2+ fluxes associated with either the acrosome reaction or sperm motility.
The specific biochemistry underlying CRISP2 function and, indeed, that of most CRISPs, is lacking. As such, the aim of the current study was to identify CRISP2 interacting proteins. To this end, a yeast two-hybrid screen of a mouse testis expression library was performed using the putative mature form of CRISP2 as bait. Using this approach and expression analysis, mitogen-activated protein kinase kinase kinase 11 (MAP3K11, previously called mixed-lineage kinase [MLK] 3) was identified as a CRISP2 interacting protein and localized to the developing acrosome of round through elongated spermatids and the acrosome of epididymal sperm. The binding of CRISP2 to a kinase is suggestive of a role for the CRISP2-MAP3K11 complex in acrosome development.
Anti-human MAP3K11 C-20 and A-20 sera and the C-20 immunizing peptide were obtained from Santa Cruz Biotechnology. The C-20 immunizing peptide was derived from the COOH end of MAP3K11, and the A-20 peptide was derived from the NH2 end. CRISP2 (T4) antiserum was as described previously [1]. His-tag (27E8) monoclonal antibody (#2366) was obtained from Cell Signaling Technology. Alexa Fluor 680-, 488-, and 546-conjugated goat anti-rabbit immunoglobulin (Ig) Gs were obtained from Molecular Probes (Invitrogen, Australia). Infrared (IR) Dye 800-conjugated goat anti-mouse IgG was obtained from Rockland, Mouse IgG1 was obtained from DAKO Cytomation (Denmark), and streptavidin-bound Texas red was obtained from Amersham Pharmacia (Sweden).
An adult mouse testis expression library was screened for CRISP2-binding proteins using the Matchmaker Gal4 Two-Hybrid System 3, per the manufacturer's instructions (Clontech). The putative mature CRISP2 coding region (amino acids 23–243) was amplified and cloned into NcoI and EcoRI sites of the pAS2–1 bait vector (sense primer: 5'-CGCGCGCGCATGCCATGGCATGTAAGGATCCAGACTTTACTTC-3' and antisense primer: 5'-CGCGCCGGAATTCCGGTCCTGCTGCACACTG-3'). Clones infecting PJ694A or AH109 yeast colonies that grew on tryptophan, leucine, histidine-deficient (TLH–) media were PCR amplified (using pACT2F: 5'-GGCCAAGATTGAAACTTAGAGG-3' and pACT2R: 5'-ATACCCCACCAAACCCAAA-3' primers) and cloned into pGEMTEasy. Clones were sequenced by the chain termination method with the use of the DyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, UK) at the Gandel Charitable Trust Sequencing Centre at the Monash Medical Centre using standard protocols. Sequences were analyzed using web-based bioinformatic tools.
Deletion Studies to Define Regions of CRISP2 and MAP3K11 Interaction
Crisp2 deletion constructs were produced to determine essential regions for CRISP2 binding with MAP3K11 and tested for binding with the A3 clone in yeast two-hybrid assay. Clone A3 encoded the C-terminal 20 amino acids of mouse MAP3K11 and was typical of those identified in the original screen. Clone A3 consisted of an 8.1-kb pACT2 backbone and a 621-bp insert, of which 63 bp encoded the C terminus of mouse MAP3K11 and the remainder encoded 3' untranslated region (UTR). Crisp2 deletion constructs were designed based on our current knowledge of CRISP function and homology to members of the CAP superfamily. Primers were designed to amplify segments of Crisp2 coding sequence with engineered restriction sites in order to directionally and in-frame clone the truncated Crisp2 deletion products into the bait vector, pAS2–1. Seven deletion constructs were produced (Fig. 1): Crisp2
1 (amino acids 79–243 of the full-length mouse protein) was amino-terminally truncated to omit the putative cell adhesion domain [12]; Crisp22 (amino acids 23–188) was carboxy-terminally truncated to remove the CRISP domain [5, 6]; Crisp23 (amino acids 189–243) contained the CRISP domain alone (composed of both the hinge and ICR regions) [5, 6]; Crisp24 (amino acids 23–154 fused to amino acids 189–243) had the midregion encoding part of the CAP signature motif removed, thereby excluding three of the six cysteines involved in intramolecular disulphide bonding within the CAP domain [6, 19]; Crisp25 (amino acids 203–243) contained the ICR of the CRISP domain; Crisp26 (amino acids 23–185 fused to amino acids 204–243) contained the entire Crisp2 sequence minus the hinge region and Crisp28 (amino acids 188–203) contained the hinge region alone [5, 6]. A standard PCR approach was used to clone deletion constructs into pAS2–1 bait vector.
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Production of Recombinant CRISP2 CRISP Domain
The CRISP2 CRISP domain (amino acids 189–243) was produced in Escherichia coli as a His-tagged fusion protein (His:CRISP2) and purified as described previously [5].
CRISP2 and MAP3K11 Pull-Down Experiments
Spermatozoa were harvested from the cauda epididymides of 10-week-old F1 (CBA x C57Bl6) males in PBS supplemented with Protease Inhibitor Cocktail Set III (Calbiochem). The sperm were pelleted by centrifugation at 3 000 rpm for 5 minutes at room temperature, and then resuspended in 10 mM PIPES, pH 6, 150 mM NaCl, and Protease Inhibitor Cocktail Set III. Sperm proteins were released by sonication for 6 x 10 seconds on ice using an Ultrasonics homogenizer 4710 series at an amplitude of 60 µm. Insoluble material was removed by centrifugation at 10 000 rpm for 2 minutes at room temperature and the soluble sperm extract supernatant used for immunoprecipitation experiments. Binding assays were performed at pH 6 based upon nuclear magnetic resonance data showing improved structural definition of the CRISP domain at this pH [5].
Approximately 100 µg of sperm extract was incubated with 1 µg His:CRISP2 CRISP domain and 108 His-tag antibody-coupled M-280 Tosylactivated Dynabeads (Dynal Biotech) in 10 mM PIPES, pH 6, 150 mM NaCl, and Protease Inhibitor Cocktail Set III, overnight at 4°C with gentle rolling. His-tag monoclonal antibody was coupled to Dynabeads according to the manufacturer's instructions. Unbound sperm proteins were removed using two 10-sec washes in ice-cold PBS. Protein complexes were eluted from the beads in 2x reducing SDS-PAGE sample buffer, resolved on 10% SDS-PAGE, and transferred onto Hybond-C Extra supported nitrocellulose membrane (GE Biosciences). Nonspecific antibody binding was minimized with Odyssey blocking buffer (Li-Cor Biosciences) and Western analysis performed using 0.2 µg/ml of the C-20 MAP3K11 antiserum and 0.2 µg/ml of Alexa Fluor 680-conjugated goat anti-rabbit IgG. His:CRISP2 CRISP domain was detected using 1 µg/ml His-tag antibody and 0.2 µg/ml IR Dye 800-conjugated goat anti-mouse IgG. Membranes were imaged and analyzed using an Odyssey Infrared Imaging System (Li-Cor Biosciences). Nonspecific MAP3K11 binding was assessed by parallel experiments using Dynabeads coupled to an irrelevant antibody of the same isotype (IgG1). Subsequent coimmunoprecipitation experiments were repeated with PBS at pH 7.0 to determine the pH dependence of the interaction between MAP3K11 and CRISP2.
Map3k11 mRNA and Protein Expression
Map3k11 mRNA expression within the testis and somatic tissues was initially determined by Northern blot hybridization, as previously described [20], with a 188-bp cDNA probe amplified from mouse testis cDNA using primers Map3k11-1 (5'-CACTGGAAGAGGAACCAGGA-3') and Map3k11-2 (5'-TAGGTACAGTGTCAGGCCCC-3'). Tissues were harvested from juvenile (Postnatal Days 0, 14, 18, 22, and 30) and adult mice (Postnatal Day 36), conducted in accordance with the National Health and Medical Research Council's Guidelines on Ethics in Animal Experimentation and approved by the Monash Medical Centre Animal Experimentation Ethics Committee. Total cellular RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi [21].
MAP3K11 protein expression within somatic tissues and testes during defined periods of postnatal development was assessed by Western blotting using the C-20 antiserum. Somatic tissues were harvested from adult F1 mice. Testes were taken from Postnatal Day-14, –22, –30, and –60 mice. Sperm were harvested from adult cauda epididymides. Protein was extracted by homogenization in RIPA buffer [22] containing protease inhibitors (Calbiochem-Novabiochem GmBH, Germany). Samples were electrophoretically separated on 10% SDS-PAGE gels and processed for Western blots, as described above. Identical preparations of epididymal sperm and testis extract were probed using the A-20 serum in order to assess the possibility of cleavage of the 93-kDa form of MAP3K11 generally seen in somatic tissues. Negative controls included the omission of the primary antiserum and preabsorption of the primary serum with the immunizing peptide.
The localization of MAP3K11 protein within testis sections and acetone-methanol-fixed cauda epididymal sperm was determined at a cellular level using the MAP3K11 antiserum, as previously described [20]. Negative controls included the omission of the primary antiserum and preabsorption of the primary serum with the immunizing peptide.
The potential for colocalization of MAP3K11 and CRISP2 in sperm was assessed using immunofluorescence. Sperm samples were air dried onto SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany). Sections were fixed in 4% paraformaldehyde in PBS for 2 min and then washed in Tris-buffered saline (TBS). Samples were permeabilized in 0.2% Triton X100 in TBS for 1 h at room temperature and then washed in TBS. Nonspecific binding was eliminated by blocking in 10% normal horse serum (NHS)/0.2% Triton X100 in CAS-BLOCK (Zymed, CA) blocking solution for 30 min at room temperature. Preparations were then incubated overnight at 4°C with the C-20 MAP3K11 diluted 1:20 in 10% NHS/TBS. After washing, the MAP3K11 antibody was detected by incubating slides for 1 h with secondary Alexa Fluor 488 goat anti-rabbit diluted 1:200 in TBS. After extensive washing, slides were reblocked in 10% NHS/PBS for 1 h at room temperature. Samples were then incubated with the T4 CRISP2 serum diluted 1:20 in TBS overnight at 4°C. After washing, CRISP2 was visualized using goat anti-rabbit Alexa Fluor 546 Ig diluted 1:200 in TBS incubated at room temperature for 1 h. DNA was visualized using 4',6'-diamidino-2-phenylindole. Slides were mounted with DAKO fluorescent mounting media and sperm immunofluorescence visualized on a fluorescent microscope (Olympus, Tokyo, Japan). Negative controls included secondary antibodies incubated in the absence of both primary antibodies, and in the absence of one primary antibody alone, to ensure that green fluorescence was specific for the MAP3K11 antiserum and red fluorescence was specific for the CRISP2 antiserum. Colocalization was evidenced by the presence of a yellow signal.
CRISP2 Is a MAP3K11-Binding Protein
A yeast two-hybrid screen of a mouse testis expression library with the putative mature form of CRISP2 identified several potential binding protein sequences. One of the most frequently identified cDNA sequences was that of mouse Map3k11. Sequencing of all eight clones revealed that they all encoded the entire 3' UTR and the 20 carboxyl-most amino acids of Map3k11 (as typified by clone A3). In order to define the region of CRISP2 that interacted with MAP3K11, Crisp2 deletion constructs were cotransfected with Map3k11 clone A3 into yeast and their ability to interact assessed on TLH– media. Cultured yeast, which was cotransfected with Map3k11 and deletion construct Crisp21, Crisp23, and Crisp24, grew on TLH– nutrient-deficient media. Each of these constructs had the full CRISP domain in common. In contrast, yeast cotransfected with Map3k11 and Crisp22, Crisp25, Crisp26, and Crisp28 were unable to grow on TLH– media. These data demonstrate that the CRISP domain of CRISP2 binds to the carboxyl terminus of MAP3K11. Within the CRISP domain, the region of interaction involved, or was influenced by, sequences in both the hinge and ICR regions (Fig. 1).
CRISP2 and MAP3K11 binding was confirmed using pull-down experiments in which sperm extracts were incubated with recombinant His:CRISP2 CRISP domain and pulled down with anti-His-tagged IgG coupled to Dynabeads. As can be seen in Figure 2, recombinant CRISP2 CRISP domain bound to an 60-kDa variant of MAP3K11 detected by Western blot. This protein was not observed when extracts were incubated in the presence of an irrelevant protein coupled to Dynabeads, thus indicating the specificity of the interaction. Similarly, the interaction was not observed at pH 7.0, suggesting that the interaction, as with the three-dimensional structure of CRISP2 CRISP domain, is influenced by the pH of the surrounding milieu [5].
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MAP3K11 Within the Testis and Epididymal Sperm
Surprisingly, the pull-down experiments identified an 60-kDa variant of MAP3K11 in epididymal sperm, rather than the 93 kDa version seen in most somatic tissues [23], as also observed by us (Fig. 3A). The MAP3K11-reactive band in the liver in Figure 3A corresponds to an unknown modification of MAP3KII resulting in the smaller 44-kDa band. This immunoreactive band is removed following preadsorption of the primary antibody with the immunizing peptide, demonstrating the specificity of the interaction. The 93-kDa form of MAP3K11 in the testis was also consistently seen by us when using the A-20 serum (directed against the NH2 terminus), but variably when using the C-20 serum (directed against the COOH terminus) (Fig. 3, B and C, respectively). Within sperm, an 60-kDa variant of MAP3K11 was reliably seen when the C-20 serum was used, whereas no bands were observed when the A-20 serum was used (Fig. 3, B and C). Within testis extracts, when using the C-20 antiserum, an 70-kDa form was also seen, in addition to the 60-kDa form. Using the A-20 antibody on testes extract, we observed the 93-kDa form, but also an 40-kDa form and an 23-kDa form (Fig. 3B). Collectively, these data suggest that the 93-kDa form of MAP3K11 is produced within the testes, but is cleaved into fragments of 70 kDa (plus 23 kDa) and 60 kDa (plus 40 kDa), but only the 60-kDa COOH-terminal fragment is incorporated into the sperm acrosome. Consistent with this hypothesis, a Western blot using the C-20 serum on testes of various ages during the establishment of full spermatogenesis (Fig. 3D) showed a progressive loss of the 93-kDa form and concomitant increase in the 70-kDa form. It is of note that the 60-kDa form appeared only after Day 30 of testis development, consistent with the appearance of elongating and elongated spermatids. Caution must be shown, however, in assigning a relative molecular weight of MAP3K11 to particular germ cell types based on these data, since Leydig cells were also immunopositive for MAP3K11, as shown in Figure 4A. The specificity of the C-20 antiserum was supported by the elimination of immunoreactive bands following preabsorption with the immunizing peptides (Fig. 3A, lower panel).
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An alternative hypothesis for the presence of the 70-kDa C-20 immunoreactive band in the testis is alternative splicing. Although we found no evidence of this based on Northern blot data using a probe directed to the 3' end of the coding region (Fig. 5), a recent entry into the mouse Expressed Sequence Tag repository indicates the presence of a novel MAP3K11 variant, which would encode a protein of 68 kDa. This sequence is typified by AAH30928 from salivary gland, and contains a partially truncated putative kinase domain.
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The observed interaction between CRISP2 and MAP3K11 would only be biologically relevant if the proteins were spatially and temporally coexpressed. In order to assess this possibility, MAP3K11 protein expression within the testis and in epididymal sperm was determined. Initially, as an indication of the testicular cell types within which MAP3K11 was produced, mRNA from testes of defined ages during development was assessed in addition to a range of somatic tissues. A single 3.8-kb Map3k11 mRNA transcript was detected in testis, epididymis, ovary, lung, kidney, spleen, stomach, large intestine, small intestine, and, to a lesser degree, in liver, brain, and heart (Fig. 5). Within the testis, Map3k11 mRNA was first detected at Day 14. Higher levels of expression were observed in Day-30 and Day-36 testes. This pattern of Map3k11 mRNA expression was strongly suggestive of production in pachytene and secondary spermatocytes and round spermatids, and an upregulation in elongating spermatids and elongated spermatozoa (Fig. 5). The pattern of mRNA expression within the testis was consistent with the immunohistochemistry data for MAP3K11 protein. Using the C-20 MAP3K11 antiserum, MAP3K11 protein was localized to the cytoplasm of late spermatocytes and round spermatids. More intense staining was seen in the region of the proacrosomal granule and acrosome of round through to elongated spermatids (Fig. 4A) in a position consistent with the expression of CRISP2 protein [1, 2, 13]. MAP3K11 protein was also detected in Leydig cells, but not in spermatogonia, early primary spermatocytes, Sertoli cells, or peritubular myoid cells. Staining specificity was assured by a lack of staining when the antiserum was preabsorbed with the immunizing peptide (Fig. 4B).
Immunofluorescence localization of MAP3K11 alone on caudal epididymal sperm showed staining on the acrosome, and was consistent with localization observed in testis (Fig. 4C). Occasionally, MAP3K11 staining was also observed within the midpiece of sperm at a light-microscopic level (Fig. 4C). Preabsorption of the MAP3K11 antiserum with the immunizing peptide completely abolished sperm immunofluorescence, confirming the specificity of the antibody recognition (Fig. 4D). Immunofluorescence was also performed to assess the potential for colocalization of MAP3K11 and CRISP2. Double labeling, as evidenced by a yellow signal, could be clearly seen in the acrosome of caudal epididymal mouse sperm, confirming the colocalization of MAP3K11 and CRISP2 in the sperm acrosome (Fig. 4E). Consistent with previous studies [1], CRISP2 labeling was also seen in the mid- and principal pieces of the sperm tail.
These studies demonstrate that MAP3K11 is a CRISP2-binding protein; that this interaction is specific to the CRISP domain of CRISP2 and the C-terminal 20 amino acids of MAP3K11, and is dependent on the pH of the surrounding milieu; that MAP3K11 is colocalized with CRISP2 in the developing and mature sperm acrosome; and that MAP3K11 appears to undergo processing during spermiogenesis to form novel 70-kDa and 60-kDa variants, of which only the 60-kDa form is found in mature sperm. These data raise the possibility that the CRISP2-MAP3K11 complex is involved in sperm acrosome development.
MAP3K11 has been most extensively characterized as a widely expressed serine/threonine MAP3K with the ability to act as an upstream activator of MAPK8 (previously called c-jun-N-terminal kinase), which, in somatic cells, becomes activated in response to cytokines and environmental stress [24–28]. More recently, MAP3K11 has also been implicated in the activation of other MAPK proteins, including MAP2K4 [29] and MAPK1 (previously known as extracellular signal-regulated kinase and p38) [27, 29], and in the activation of the structurally unrelated pathway involving NFKB [30]. In all of these signaling events, activation of MAP3K11 affects diverse cellular processes through transcriptional change. However, within elongate spermatids, within the acrosome compartment, MAP3K11 is likely involved in the phosphorylation of target proteins associated with sperm acrosome development. Such a role would be analogous to MAP3K11's role in phosphorylating Golgi autoantigen, golgin subfamily a, 3 (Golgin-160) prior to apoptosis in HeLa cells [31]. An interaction between CRISP2 and a kinase also raises the possibility that MAP3K11 acts to directly phosphorylate CRISP2. In support of this hypothesis, a predicted phosphorylation site exists within the CRISP2 CRISP domain at position S212, a position consistent with the site of MAP3K11-CRISP2 interaction defined herein. Such a possibility is the subject of ongoing investigations.
Structurally, MAP3K11 contains several features in common with members of the MLK family: an src homology 3 (SH3) domain, a serine/threonine and tyrosine kinase catalytic domain, two tandem leucine zippers, a basic domain, a Cdc42/Rac interactive binding motif, and a C-terminal extension rich in proline, serine, and threonine [23, 25]. MAP3K11 is unique, however, in that the carboxyl terminus extends approximately 320 amino acids further compared with other MLK family members. This extension includes the entire proline-rich region, which has been shown to undergo several serine phosphorylations [32] mediated by MAPK8, and which controls localization to Triton X100-soluble and -insoluble membrane domains in somatic cells [33]. It is to the extreme end of this unique region that CRISP2 binds, thus making it unlikely that other members of the MLK family will bind to CRISP2 in the same manner. Such proline-rich regions have frequently been characterized as protein interaction domains [34, 35].
The detection of novel 70-kDa and 60-kDa forms of MAP3K11 within the testis and spermatozoa was unexpected. Western blot data suggest that they are proteolytic cleavage products of the 93-kDa form of MAP3K11 observed in most tissues, including the testis. The point of cleavage and the effect this will have on kinase activity is currently unknown. Based on what is known of the structural elements of MAP3K11 and the C-20 and A-20 epitopes, we predict that cleavage of the 93-kDa form to the 70-kDa form in the testis would remove all of the SH3 domain and a portion of the tyrosine kinase domain; however, the catalytic core of Thr277/Ser281 would remain intact. Further truncation to the 60-kDa form in epididymal sperm would truncate the N terminus, removing the catalytic core of the kinase domain, and ablate kinase activity. Cleavage may therefore be a regulatory mechanism employed within the acrosomal compartment to regulate MAP3K11 activity. Interestingly, the cone snail CAP protein, TEX31, which is structurally related to CRISP2, has been characterized as a site-specific protease [36], thus raising the possibility that CRISP2 cleaves MAP3K11 as a means of regulating its function.
The interaction between CRISP2 and MAP3K11 only at acidic pHs is also of interest. Studies from several species have shown that the pH of the acrosome is acidic, and that it undergoes alkalinization during capacitation [37–40]. For example, Nakanishi and colleagues [37] used an enhanced green fluorescent protein acrosome mouse model to calculate that mouse sperm acrosomal pH increases from pH 5.3 to pH 6.2 during capacitation. Comparable studies in the rat have shown an increase in pH from 6.54 to 6.73 [38]. In addition, previously published work shows that the structure of the acrosomal contents is also affected by pH. Specifically, the acrosomal matrix remained intact at acid pHs, but dissolved, through the activity of a trypsin-like protease, at pH 7 and higher [37, 41]. Whether the MAP3K11-CRISP2 complex forms part of the acrosomal matrix remains to be conclusively determined, but the concept is supported by recent selective solubilization experiments for CRISP2 performed on mouse sperm [14].
It is of note that a Map3k11 knockout mouse line has been produced [42]. Null mice have subtly abnormal tumor necrosis factor regulation, but have apparently normal male fertility. This raises several possibilities: 1) MAP3K11 is obsolete within spermatogenesis; 2) its function may be compensated for by other kinases; or 3) knockout mice have a subtle reproductive phenotype, which may only become evident upon closer examination. The latter possibility is most well illustrated in the instance of the Nos2 null mouse line, whereby null males produce approximately 65% more sperm per day than wild-type litter mates [43].
Previous to our study, the only biochemical pathway to which CRISP2 had been shown to have a function was with RYRs, where the CRISP domain has been shown to differentially regulate Ca2+ gating. Based on the data outlined herein, we propose that the CRISP2-MAP3K11 complex has a role in acrosome development within the testis. The data described herein further define the CRISP domain, composed of both the hinge and ICR regions, as a MAP3K11 interacting domain and defines a novel function for the unique carboxyl extension for MAP3K11.
ACKNOWLEDGMENTS
Thanks go to Stephanie Smith and Amanda Harman for technical assistance.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported in part by funding from National Health and Medical Research Council grant 334011, the New South Wales Department of State and Regional Planning, and Australian Research Council grant CEO348239 to M.K.O.B., R.J.A., and D.M.D.K.
Correspondence: 2Correspondence: Moira O'Bryan, Monash Institute of Medical Research, Monash University, 27–31 Wright St., Clayton 3168, VIC, Australia. FAX: 61 3 9594 7439; e-mail: moira.obryan{at}med.monash.edu.au
Received: 5 September 2006.
First decision: 27 October 2006.
Accepted: 20 March 2007.
REFERENCES
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