Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth.
The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R.E. Wilson et al., Cancer Res., 49:711-716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 +/- 50 pmol/min/mg SD) compared with growing cells (22.8 +/- 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 +/- 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Mel-ab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 +/- 147 cpm/10(6) cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 +/- 28 and 947 +/- 81 cpm/10(6) cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.
Brooks
G
G
Biology of Metastasis Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Wilson
R E
RE
Dooley
T P
TP
Goss
M W
MW
Hart
I R
IR
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004789:Enzyme Activation; D007700:Kinetics; D008544:Melanocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D008546:Melanoma, Experimental / Q000201:enzymology / Q000473:pathology; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010750:Phosphoproteins; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate