Nov 1, 1989 - also reacted with this antiserum. Antiserum to bFGF detected a 90 kDa protein component in the membranes and in the nuclear sediments from ...
494 also reacted with this antiserum. Antiserum to bFGF detected a 90 kDa protein component in the membranes and in the nuclear sediments from the highly metastatic RMSO cells. A correlation seems to exist in this model between the expression of the metastatic phenotype and the expression of a 90 kDa protein containing a basic fibroblast growth factor epitope. This work was supported by the Association pour la Recherche sur le Cancer. 1. Biswas, C. ( 1988)J. Cell. Physiol. 136, 147- 153 2. Redini, F., Moczar, E., Antoine, E. & Poupon, M. F. (1989) Riochcm. Biophys. Actu. 991,359-366
BIOCHEMICAL SOCIETY TRANSACTIONS 3. Wright, T. C., Johnstone, T. V., Castellot, J. J. & Karnovsky, M. J. (1985)J. Cell. Physiol. 125,499-506 4. Sweeney, F. L., Pot-Deprun, S., Poupon, M. F. & Chouroulnikov, I. (1980) Cancer Res. 42,3775-3782 5. Underhill, C. B., Chi-Rosso, G. & Toole, B. P. ( 1 983) J. Biol. Chem. 258,8086-8091 6. Hollenberger, M. D. & Cuatrecasas, P.(1979)in The Receptors (O'Brien, R. D. ed). vol. 1 Plenum Press, New York 7. King, J. & Laemmli, U. K. (1971)J. Mol. Biol. 62,465-473
Received 2 1 November 1989
Phorbol ester-induced downregulation of protein kinase C potentiates insulin receptor tyrosine autophosphorylation: evidence for a major constitutive role in insulin receptor regulation immunoblotting 191. '2sI-Labelled insulin binding was unchanged in response to acute or chronic PMA treatment. The effect of PKC downregulation to potentiate insulinstimulated tyrosine kinase activity was unexpected in view of the apparent lack of effect on receptor function after acute activation of PKC and the greater increase in serine/ threonine phosphorylation with chronic PMA treatment. It must be assumed that downregulated cells possessed residual PKC activity which escaped immunodetection o r that more than one serine/threonine kinase acts on the receptor. It remains possible that the observed receptor phosphorylation Insulin-stimulated receptor autophosphorylation and kinase is not directly catalysed by PKC itself, but involves an interactivation are thought to be essential for its signal transduc- mediate kinase. tion [ 11. Phorbol ester-induced receptor phosphorylation It is clear that the level of serine/threonine phosphorylaattenuates its tyrosine kinase activity in some cell types [2,3]. tion of receptor in these cells is not inversely correlated with In others, the effects are unclear in that the phorbol ester- tyrosine kinase activity, as has been reported in other cells [2, induced phosphorylation appears to be additive with insulin 31. It is possible that the sites and consequences of serine/ threonine phosphorylation are different in different cell [4,51. We investigated the effects of 4-B-phorbol 12-myristate types, perhaps reflecting the activity of specific PKC 13-acetate (PMA) on receptor phosphorylation in a cell line isoforms. Alternatively, the serine/threonine phosphoryla(NIH-3T3 HIR 3.5) that over-expresses human insulin tions may occur on separate receptor subpopulations, as has receptor cDNA [6].Cells labelled with j2P for 2 h were either been suggested to occur in other cells [lo]. This could be pre-exposed to PMA for 12 h or incubated with PMA for 30 investigated using anti-phosphotyrosine rather than antimin. Both groups were then exposed to insulin for 5 min, receptor antibodies for immunoprecipitation. The mechanism whereby PKC inhibits insulin-stimulated solubilized and the receptor immunoprecipitated [7]. Labelled B-subunit on SDS/polyacrylamide gels was receptor phosphorylation, as manifest in the potentiation of identified by autoradiography and subjected to phospho- insulin response in PKC-downregulated cells is unclear. It is amino acid analysis and tryptic phosphopeptide mapping [8]. difficult to account for this by the low level of basal (no Acute exposure to phorbol esters stimulated the phos- insulin or PMA) receptor serine/threonine phosphorylation phorylation of the insulin receptor by 3-4-fold on sites which is observed by "P-phosphate labelling, although the contained in five tryptic phosphopeptides. The phorbol presence of phosphate which- turns over slowly and is not ester-induced phosphorylation was additive with the insulin- labelled under the conditions of these experiments cannot be stimulated tyrosine autophosphorylation and no inhibition ruled out. Alternatively, PKC may act on the receptor by was seen using several assays to measure insulin-stimulated mechanisms other than direct phosphorylation. tyrosine autophosphorylation and kinase activity (not It has been reported that chronic exposure to high concentrations of insulin induces a refractory state in which shown). In contrast, chronic pre-exposure to phorbol esters receptor kinase activity is decreased without accompanying enhanced the insulin-stimulated tyrosine phosphorylation by increase in serine/threonine phosphorylation [ 1 11. It is 2-fold at both maximal and submaximal concentrations (Fig. interesting to speculate that the mechanism of this effect is 1). This occurred in spite of the fact that cells exposed to related to the tonic inhibition exerted basally by PKC, as PMA for 12 h showed even greater serine/threonine observed here. However, details of these regulatory phosphorylation of receptor than when PMA was added mechanisms remain to be elucidated. acutely (30 min), on the same set of tryptic phosphopeptides. T.S.P. was the recipient of the Emanuel Bradlow Scholarship However, chronic PMA treatment caused protein kinase C (PKC) downregulation as evidenced by its absence on from the Bradlow Foundation, South Africa and St John's College, T. S. PILLAY,$ JONATHAN WHITTAKER? and KENNETH SIDDLE* *Department of Clinical Biochemistry, University of Cambridge, Addenbrooke 's Hospital, Hills Road, Cambridge CB2 2QR, U.K . ? Division of Endocrinology, Department of Medicine, Health Sciences Center, SUNY at Stony Brook, Stony Brook, NY I1 794 U.S.A.and $Division of Molecular Endocrinology, Department of Chemical Pathology, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN U.K .
Abbreviations used: PMA, 4-/3-phorbol 12-myristate 13-acetate; PKC, protein kinase C.
Cambridge. K.S. was a Wellcome Senior Lecturer. This work was supported by the Wellcome Trust, the M.R.C., the Juvenile Diabetes Foundation and by a grant from the Queen Charlotte's and Hammersmith Special Health Authority to T.S.P.
1990
633rd MEETING, LONDON
495
(a)
---Subunit
log{[lnsulin] (M))
log{(lnsulin] (M))
-
-11
-
-11 -10 -9 -8
-10 - 9
-8
.-11 -10 -9
-7
-7
-
-11
-10
-8
-9
-7
-8
-7
w PMA ( 1
,UM)
Fig. 1. Eflects of' chronic exposure 10 phorbol esters on insulin receptor phosphorylution (a) Dose-response of insulin-stimulated phosphorylation in control cells and cells preincubated with 1 ~ M - P M Afor 12 h. (b) Phosphoamino acid analysis of the labelled /I-subunits shown in (a). Abbreviations: PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine. I . Houslay, M. D. & Siddle. K. ( 1989) Hr. Med. Hull. 45,264-284 2. Takayama, S., White, M. F., Lauris, V. & Kahn, C. R. (1984) I'roc. Nutl. Acud. Sci. U.S.A. 81,7797-7801 3. Takayama, S., White, M. F. & Kahn, C. R. ( 1988) J. Biol. Chem. 263, 3440-3447 4. Jacobs. S., Sahyoun, N. E., Saltiel, A. R. & Cuatrecasas, P. (1983) I'roc. Nurl. Acud. Sci. U.S.A. 80,621 1-6213 5. Jacobs, S. & Cuatrecasas, P. (1986) J . Biol. C'hem. 261. 934-039 6. Whittaker. J., Okamoto. A. K.. Thys, R., Bell, G . I.. Steiner, D. F. & Hofmann. C. A. (1987) Proc. Nurl. Acud. Sci. U.S.A. 84, 5237-5241
7. Soos, M. A,, O'Brien, R. M., Brindle, N. P. J., Stigter, J. M., Okamoto, A. K., Whittaker, J . & Siddle, K. (1989) Proc. Nutl. Acad. Sci. U.S.A. 86,5217-5221 8 . Tavare, J. M., OBrien, R. M., Siddle, K. & Denton, R. M. (1988) Eiochem. J. 253,783-788 9. Adams, J. C. & Gullick, W. J . ( 1 989) Eiochem. J. 257,905-91 1 10. Brillon, D. J., Friedenberg, G. R., Henry, R. R. & Olefsky, J . M. (1989) Diabetes 38,397-403 I I . Treadway,J. L., Whittaker, J. & Pessin, J. ( I 989) J. Biol. Chem. 264,15136-15143 Received 23 November 1989
Heparin-binding proteins from human melanoma cells JOSEF TIMAR,* ANDREA LADANYI,* MADELEINE MOCZAR,? KAROLY LAPIS* and ELEMER MOCZARt tlahoratoire de Biochimie du Tissu Conjonctij 8 rue du GPnPrul Surrail, 94010, CrPteil, France and'*lnstitute of Puthology and Cancer Research, Ulloi ut 26, 1084, Budapest, I i11ngury Several studies implicate glycosaminoglycans in the regulation of cell behaviour. This function can be accomplished either locally by direct contact of polyoside sequences with surface receptors o r distally after their proteolytic or endoglycosidasic cleavage of biologically active fragments of
Vol. 18
the molecules [ 11. Glycosaminoglycan-binding sites were observed in different malignant cell types as B 16 melanoma cells 121, Lewis lung tumor cells [3] and rat rhabdomyosarcoma cells [4, 51. The decreased expression of glycosaminoglycan-binding proteins in rhabdomyosarcoma cells seemed to be related to the decreased number of receptor sites for iduronate-containing glycosaminoglycans 141 and for hyaluronate [ 5 ] . In the present work, the heparin-binding proteins were studied in human melanoma cells in culture. Cells were injected intrasplenically in mice and the variants with decreasing metastatic capacity were selected from liver metastases. Melanoma cell variants M1, HT 168 and H T 1 8
