| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The protein kinase C (PKC) family of serine/threonine protein kinases plays critical roles in many signal-transducing pathways in mammalian cells. Twelve distinct PKC isozymes have been identified in the cells. Rat pituitary GH cells express PKC transcripts for PKCα,βII,δ,ε,η,and ζ. Among them, PKCε is strongly activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary and neuronal function, and regulates various functions including prolactin secretion. To determine the identity of components in the pathway downstream of PKCε activation, we analyzed for phosphoproteins in GH cells. The exposure of GH cells to TRH or TPA increased the phosphorylation of MARCKS, stathmin, and keratin (K), and decreased the phosphorylation of cofilin and desmin, as assessed by 2D-gel electrophoresis, amino acid sequencing, and immunological analysis. Phosphorylation of MARCKS and K was enhanced in PKCε-overexpressing GH cells, and purified PKCε directly phosphorylated both of these proteins in in vitro experiments. Mass spectrometric analysis of the endogeneos K and the synthesized peptide demonstrated that at least three seine residues of K were preferentially phosphorylated by PKCε. Moreover, TRH-mediated physiological responses were enhanced in the PKCε-overexpressing cells. These data suggest that K is a specific substrate for PKCε and a mediator of TRH-PKCε signaling in GH cells.
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