Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy. Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases. Both genes were intronless and mapped to chromosomes 5 and 22 respectively. The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies. Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis. However, mRNA for these kinases can be detected in other tissues using real-time PCR. In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned. TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR. Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation. TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro. Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm. In contrast, TSKS was only found in the testis. The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca2+) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca2+ modulates this process has not yet been completely characterized. Ca2+ can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca2+/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca2+ in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca2+ regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

The molecular basis of mammalian sperm capacitation is unique in that, it is associated with a protein kinase A (PKA) dependent upregulation of protein tyrosine phosphorylation. Therefore, PKA activity during capacitation would be crucial for the downstream events of protein tyrosine phosphorylation, and mechanisms may exist to ensure that PKA phosphorylates its specific substrate. This could be achieved by bringing PKA close to its substrate, a function normally carried out by an A-kinase anchoring protein (AKAP). We showed previously that cauda epididymidal spermatozoa of hamster undergo a capacitation-dependent increase in protein tyrosine phosphorylation. In the present study, evidence is provided that two major tyrosine phosphorylated proteins of molecular weight 97 and 83 kDa are the hamster homologues of mouse pro-AKAP82 and AKAP82, and have been designated as hamster pro-AKAP83 and AKAP83 respectively. Hamster AKAP83 resembled the mouse AKAP82 with respect to its molecular weight, pI (pH 5-5.5) and cDNA and amino acid sequences. Sequence analysis indicated that the primary structure of pro-AKAP83 was highly conserved and exhibited 91% identity with mouse and rat AKAP82. Further, the functional domains, namely the region involved in binding the regulatory subunit of PKA and the proteolytic cleavage site between pro-AKAP83 and AKAP83, were identical with that observed in rat and mouse pro-AKAP82 and AKAP82. Immunoblot analysis using polyclonal hamster anti-AKAP83 antibodies indicated that AKAP83 was present both in caput and cauda epididymidal spermatozoa. The antibody also identified the pro-AKAP82 and AKAP82 in mouse caput and cauda epididymidal spermatozoa. Immunofluorescence studies indicated that AKAP83 in hamster spermatozoa was localized along the length of principal piece of the tail. It was also demonstrated that hamster pro-AKAP83/AKAP83 gene expression was testis specific and was not expressed in other organs in either sex. This is the first report implicating AKAP in capacitation in rodents.