Protein kinase C epsilon phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone.
Protein kinase C epsilon (PKCepsilon) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH(4)C(1) cells. We analyzed the downstream mechanism after PKCepsilon activation. Exposure of GH(4)C(1) cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKCepsilon-overexpressing clones, and purified recombinant PKCepsilon directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKCepsilon and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell-cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1-53) showed that PKCepsilon phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKCepsilon-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKCepsilon, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH-PKCepsilon signaling in pituitary cells.
Akita
Yoshiko
Y
Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, Japan. akita@rinshoken.or.jp
Kawasaki
Hiroshi
H
Imajoh-Ohmi
Shinobu
S
Fukuda
Hiroyuki
H
Ohno
Shigeo
S
Hirano
Hisashi
H
Ono
Yoshitaka
Y
Yonekawa
Hiromich
H
D000595:Amino Acid Sequence; D000818:Animals; D051338:Cofilin 1 / Q000378:metabolism; D051345:Destrin / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D053533:Keratin-8 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010902:Pituitary Gland / Q000378:metabolism; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000502:physiology*; D051381:Rats; D012694:Serine / Q000737:chemistry*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D051338:Cofilin 1; D051345:Destrin; D053533:Keratin-8; D012694:Serine; D013973:Thyrotropin-Releasing Hormone; D051744:Protein Kinase C-epsilon