Abstract
Transition protein 2 (TP2), which is expressed during stages 12-15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-Calpha1 antibodies revealed that Calpha1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY375243
Because the phosphorylation sites happen to be near the nuclear localization signal of Transition protein 2 (TP2), we examined the effect of phosphorylation on its import into the nucleus and found that the transport is modulated by phosphorylation but is not required for its import into the nucleus
Phosphorylation of TP2 by Round Spermatid Cytosol—We have shown previously that rat TP2 is phosphorylated in vivo by metabolic labeling with [32P]orthophosphate and in vitro by the protein kinase A present in the high salt extract of sonication-resistant spermatid nuclei (8)
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY375243. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA).
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