A key role for a 145-kDa cytosolic protein in the stimulation of Ca(2+)-dependent secretion by protein kinase C.
Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145.
Nishizaki
T
T
Department of Zoology, University of Wisconsin, Madison 53706.
Walent
J H
JH
Kowalchyk
J A
JA
Martin
T F
TF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002850:Chromatography, Gel; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D007074:Immunoglobulin G; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine