ABSTRACT
We describe the characterization of several transcripts of the Drosophila serine/threonine protein kinase 61 (Dstpk61) gene. Dstpk61 produces at least four transcripts, including a 3.0-kb testis-specific transcript, a 4.5-kb female-specific carcass transcript, a 3.5-kb ovary-specific transcript, and a 4.7-kb non-sex-specific transcript. Two cDNAs, a 4.5-kb cDNA (cDNAB) and a 3.0-kb cDNA (cDNAA), likely to correspond to either the non-specific or the female-specific carcass and the testis-specific transcript, respectively, were fully sequenced and found to encode a novel OPA-repeat-containing serine/threonine-specific protein kinase. cDNAA and cDNAB both contain the entire ORF that encodes this predicted protein, but differ in the untranslated regions. The cDNAs contain translational control elements which are found in transcripts under male germline-specific translational control, and doublesex-like 13-nucleotide repeat elements, which are required for transformer/transformer-2-mediated splicing of the female doublesex transcript. The complex tissue and sex-specific transcripts, differing in the untranslated regions which are likely to be crucial in translational control, suggest that this kinase may have both general and sex-specific functions. The protein is homologous to human 3-phosphoinositide dependent protein kinase, which is involved in transduction of insulin signalling.
Subject(s)
Drosophila melanogaster/genetics , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , 3-Phosphoinositide-Dependent Protein Kinases , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Drosophila Proteins , Female , Male , Molecular Sequence Data , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Sex FactorsABSTRACT
BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.
