Here, we describe the identification, structure, and function of spe-8, and show that it is localized to the spermatid cell membrane but enters the cytoplasm during activation. spe-8 was the first of the five genes of the spe-8 group (spe-8, -12, -19, -27, and -29) to be identified by mutation. In contrast to the other four genes, whose sequences reveal little about their activity, the SPE-8 sequence suggests it belongs to one of the most prominent families of signaling molecules, the protein tyrosine kinases (PTKs) . These proteins route information through cells by phosphorylating specific tyrosine residues on their substrate proteins, thereby altering their substrates' chemical activities and/or binding properties. The Src homology 2 (SH2) domain, a compact domain that recognizes and binds to specific phosphotyrosine-containing proteins, is a common protein module that is often associated with PTKs and is present in SPE-8. Beyond the strong sequence similarity to these two domains exhibited by SPE-8, the fact that all six of the identified mutations lie in conserved residues of the SH2 or PTK domain underscores the importance of these domains in SPE-8 function.
PTKs belong to two general classes, the receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) . Whereas RTKs are typically bound to the plasma membrane through their own N-terminal structures, NRTKs may associate with membranes through associations of their N-terminal domains with co-receptor proteins. Based on overall sequence similarity, SPE-8 is most closely related to the Fer and Fes family of NRTKs . Human Fes has both oncogenic and tumor suppressor roles, depending upon the type of tumor [14, 25], and Fer is widely expressed and activated by growth factors, primarily platelet derived growth factor (PDGF) . Fer is involved in many processes including cell proliferation [27, 28], cell adhesion , cell migration , and even oocyte maturation . Fer is also implicated in several cancers [32–34]. Interestingly, a testis-specific alternatively-spliced isoform of Fer, FerT, has a distinct, shorter N-terminus than the full-length Fer protein and participates in acrosome formation through phosphorylation of actin remodeling proteins .
Both Fer and Fes have, in addition to their SH2 and kinase domains, several coiled-coil domains in their N-termini, which appear responsible for activation by oligomerization and subsequent trans-autophosphorylation of these proteins [18, 24]. NRTKs in general are found to localize in variable fashions either in the cytosol or bound to interior membranes via protein complexes . The SPE-8 N-terminus is unique compared to all other members of the sperm expressed NRTK family in C. elegans. It is unclear if the N-terminus of SPE-8 is responsible for formation of dimers or oligomers, or if autophosphorylation occurs. However, we speculate that this structure may be responsible for protein-protein interactions, especially given its proline-rich region, many of which are implicated in protein-protein docking or in binding SH3 domains [20, 36, 37]. Perhaps the failure of our N-terminal GFP fusion to rescue the spe-8(hc50) mutation reflects the importance of the N-terminus in SPE-8 function. Unlike the unique N-terminus, the SPE-8 C-terminus matches those of some of the family members.
Whatever the function of the C- and N-terminal domains, it is clear that interactions among the SPE-8 group proteins act to localize or stabilize SPE-8 at the plasma membrane. Localization was disrupted in mutant backgrounds for spe-12, spe-19, and spe-27, and perhaps for spe-29. It is not surprising that SPE-12 and SPE-19 proteins are required for proper SPE-8 localization because both have transmembrane domains, and SPE-12 has been shown experimentally to reside in the cell membrane [5, 7]. However, the requirement of SPE-27 for proper SPE-8 localization is unexpected, given that SPE-27 is predicted to be cytosolic . These results suggest SPE-27 may also be located at the plasma membrane. For SPE-29, the results were not clear. SPE-8 appeared to localize to the membrane in a mutant spe-29 background, but seemingly not as intensely as in a wild-type genomic background.
While SPE-8 associates with the membrane in developing sperm, its localization changes once the cells activate to spermatozoa. The protein appears to leave the membrane and become cytoplasmic in natively activated spermatozoa originating from both males and hermaphrodites. In both cases, the sperm are exposed to extracellular zinc, which acts through the SPE-8 group proteins to induce activation . We have no method for observing SPE-8::GFP localization in sperm activated only through the TRY-5 pathway. It is not possible to activate sperm in vitro with TRY-5, and spe-8 group mutant male sperm are activated only by TRY-5, but SPE-8::GFP is mislocalized due to the spe-8 group mutation. In any case, SPE-8 is dispensable for TRY-5 activation. Not only does TRY-5 activation occur in spe-8 mutant backgrounds, but SPE-8 is also mislocalized in spe-12, spe-19, and spe-27 mutant backgrounds.
It is tempting to speculate as to the phosphorylation target of SPE-8. One possible target is SPE-6. SPE-6 has a predicted role maintaining the spermatid state by inhibiting activation until the SPE-8 group signal is transduced into the cell . One hypothesis is that SPE-8 downregulates SPE-6 through tyrosine phosphorylation. SPE-6 protein exists in a perinuclear halo in spermatids (Jackson Peterson and Diane Shakes, personal communication), so the translocation of SPE-8 from the plasmalemma into the cytosol might enable interaction between the two proteins. Indeed, the NetPhos 2.0 Server  identifies four of the tyrosine residues in SPE-6 as high probability candidates for phosphorylation (data not shown). Another possible target is a C. elegans homolog of MPOP, the major sperm protein polymerization organizing protein identified in Ascaris suum
[41, 42], although a C. elegans homolog has yet to be found. MPOP is an integral membrane protein distributed throughout the plasmalemma, but it is phosphorylated by a tyrosine kinase only on the leading edge of the pseudopod, where it nucleates MSP assembly to drive pseudopodial motility. However, SPE-8 is not likely the tyrosine kinase responsible for a putative C. elegans MPOP phosphorylation because SPE-8 appears restricted to the cell body in active spermatozoa (Figure 6).
SPE-8 shares both primary sequence and protein domain architecture similarity with at least 33 other predicted paralogs in C. elegans, 29 of which have been shown to be upregulated during spermatogenesis in at least one of two studies. We include genes identified as sperm-expressed in either study because each study has slight limitations: (i) the microarray studies [9, 10] did not include all genes in the genome and may suffer from cross-hybridization, and (ii) the RNA deep sequencing study  may miss early acting sperm genes expressed whose transcripts are degraded prior to cellular encapsulation at the primary spermatocyte stage. While the majority of these genes have not received experimental attention, 30 have knockout alleles available. Only four of those knockouts produced a sterile phenotype, with the rest having no phenotype (Additional file 2). RNAi reports suggest that some of the paralogs have somatic function, although even spe-8 has an RNAi phenotype of hypersensitivity to hypoxia . Whether or not these paralogs have significant function in sperm remains unclear, but the fact that so few have phenotypes for the knockout alleles suggests that they have redundant function. The fact that so many kinases appear upregulated in sperm indicates that protein phosphorylation is a prominent regulatory mechanism during spermatogenesis. Such a conclusion is not surprising since (i) post-translational modification is the major regulatory mechanism available, owing to the cessation of gene expression at the spermatid stage, and (ii) protein phosphorylation is rapid, befitting the swift morphological transformations that take place during spermiogenesis.
Extracellular zinc is the signal for sperm activation transduced through the SPE-8 complex . The zinc signal is transmitted across the membrane to SPE-8 tethered just inside the membrane. It is unclear how reception of the zinc signal releases SPE-8 from the membrane. Zinc is an important signaling molecule in numerous cell types. For instance, the activity of most cell types in the mammalian immune system is regulated by zinc . Zinc alters sperm activity across many species, but its effect can be either positive or negative, depending upon the species (reviewed in ). Perhaps a more relevant example is the activation of p70 S6 kinase by extracellular zinc signaling in the progression of the cell cycle, with the possible involvement of intermediate NRTKs . While the identities of all intermediate factors and their specific interactions are not known, extracellular zinc induced signaling involving kinases is widespread, and our work on SPE-8 helps in understanding how spatial localization is involved in the signaling events.