Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.Erythropoietin (Epo) 1 is required for the development and maturation of erythroid cells and acts to stimulate the proliferation and differentiation of erythroid progenitor cells. Mice lacking expression of erythropoietin or its receptor die in utero due to insufficient erythropoiesis in the fetal liver (1). Erythropoietin production can be induced by hypoxia and provides physiologic regulation of the red cell mass. Erythropoietin receptor is a member of the cytokine receptor superfamily characterized by a single transmembrane domain, homology in the extracellular domain that includes a WSXWS motif, and a cytoplasmic domain that does not contain a kinase motif. Binding of erythropoietin to its receptor results in receptor dimerization, increased affinity for Jak2 to the receptor's membrane proximal region, and subsequent phosphorylation of Jak2 and tyrosines on the cytoplasmic region of the receptor (2). As with other members of this superfamily such as thrombopoietin, interleukin-3, granulocyte-macrophage colony-stimulating factor, and prolactin, Jak2 is required for signaling (3, 4) and Jak2 phosphorylation activates Stat5 (5, 6) and other signal transduction pathways. A role for calcium has been implicated in erythropoietin activity. For example, in erythroid progenitor cells, erythropoietin activates an increase in intracellular calcium in a dose-dependent manner mediated via tyrosine phosphorylation of the erythropoietin receptor requiring the cytoplasmic tyrosine 4...

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.

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We report characterization of a novel testis-and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.Hyperactivated motility along with changes in the sperm head that confer the capacity to fertilize an oocyte result from a series of time-dependent processes collectively referred to as capacitation (1, 2). Although the molecular mechanisms underlying capacitation, including hyperactivation, are incompletely understood, some fundamental processes related to capacitation have been elucidated.The presence of a protein source such as albumin, bicarbonate, and Ca 2ϩ and an energy substrate such as glucose, pyruvate, or lactate are essential to achieve in vitro capacitation (3). Capacitation is also marked by an increase in tyrosine phosphorylation through a unique signal transduction cascade involving a sperm-specific soluble adenylyl cyclase, protein kinase A (PKA), 3 and tyrosine kinase(s). A variety of factors, including Ca 2ϩ, HCO 3 Ϫ , and H 2 O 2 stimulate soluble adenylyl cyclase leading to increased cytosolic levels of cAMP (4). This increase in cAMP then activates PKA, and the consequence is a significant increase in tyrosine phosphorylation of protein substrates localized in the flagellum such as AKAP3, AKAP4, CABYR, hsp-90, ODF2, and tubulin (3,(5)(6)(7)(8)(9)(10)(11)(12).The fact that PKA is a key regulator of capacitation-associated changes such as protein phosphorylation and hyperactivation has been well established. Pharmacological stimulants that elevate intracellular cAMP, such as the phosphodiesterase inhibitors, caffeine, and pentoxifylline, enhance hyperactivated motility of sperm (13). cAMP agonists can accelerate protein tyrosine phosphorylation in sperm, whereas antagonists of PKA inhibit tyrosine phosphorylation and capacitation (14). Mice in which sperm-specific PKA ␣ is knocked out are infertile, owing to the complete absence of normal sperm movement...

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 & 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of TSSK 1 & 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.