Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.
TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.
Kiley
S C
SC
W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946-1099.
Parker
P J
PJ
Fabbro
D
D
Jaken
S
S
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000201:enzymology*; D003902:Detergents; D004789:Enzyme Activation; D005089:Exocytosis / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017830:Octoxynol; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011092:Polyethylene Glycols; D011188:Potassium / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D000199:Actins; D003902:Detergents; D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; D011092:Polyethylene Glycols; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D017830:Octoxynol; D011493:Protein Kinase C; D011188:Potassium