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Availability of PDGF receptor to PDGF stimulated tyrosine phosphorylation in mouse fibroblasts in different states of growth
164
Cell Biology International Reports, Vol. 10, No. 3, March 1986
AVAILABILITY OF PDGF RECEPTOR TO PDGF STIMULATED TYROSINE PHOSPHORYLATION IN MOUSE FIBROBLASTS IN DIFFERENT STATES OF GROWTH E. Sturani, R. Zippel, L. Toschi, L. Alberghina Dipartimento di Fisiologia e Biochimica Generali, UniversitY, Via Celoria, 26, Milano. Antibodies against phosphotyrosine (P-tyr)* (I) have allowed us to detect proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to PDGF. Western blotting of SDS PAGE fractionated proteins followed by decoration with P-tyr antibodies and 125I-protein A has been used (Zippel et al. Submitted for publication). The major tyrosine phosphorylated component appears to be the PDGF receptor in its tyrosine phosphorylated form as suggested by its molecular weight of 170 Kd (2~3), by the fact that it is detectable only in cell lines bearing the PDGF receptor (Swiss 3T~ NR6 and not in A431), and only in PDGF stimulated cells. At 37°C the phosphorylation is very rapid; the intensity of the 170 Kd band recognized by P-tyr antibodies is maximal after 5-10 min, afterwards it decreases (see also 2-3). The decrement seems not to be necessarily connected with internalization nor due to lysosomal degradation, since inhibitors of membrane internalization and of lysosomal proteolysis do not prevent the decrement of the band, which thus seems due to dephosphorylation rather than to proteolysis. This conclusion is supported by the finding that a shortage in ATP supply few min after PDGF stimulation makes the decrement more rapid, suggesting in addition that the overall phosphorylation is the net result of phosphorylation and dephosphorylation. At low temperature phosphorylation is slower but dephosphorylation (and internalization) de not take place. The PDGF concentration required for half maximal stimulation of receptor phosphorylation is about 0.5 nM (2) and is very close to that required to
induce competence acquisition during a two hours exposure to the factor. This concentration is 10-20 times higher than that required to allow entrance in S phase during a 24 hr exposure to PDGF and platelet poor plasma (4). Serum (10%-20%) stimulation, which allows S entrance, does not induce receptor phosphorylation during a 10 min to 6 hr exposure at 37°C, at least at the level of sen sitivity of our protocol. However stimulation with serum for long time (4 hrs) at 5°C allows the accumulation of the phospho rylated receptor. Thus presumably low PDGF concentrations which allow S entrance when left for a long time in contact with the cells, induce at 37°C the phosphorylation of a limited number of receptor molecules; since however the dephosphorylation is very rapid, the amount of the phosphorylated re ceptor remains at a very low steady state level, below the sensitivity of our method. 10% serum at 37°C causes downregulation of the receptor since in about 8 hr it removes from the surface of the quiescent cells the PDGF receptors as detectable by PDGF stimulated receptor phosphorylation (5). The same effect is also obtained treating the cells with PDGF and the larger is the PDGF concentration the higher becomes the rate of receptor removing. Moreover the PDGF r~ ceptor, as detected by PDGF stimulated receptor phosphorylation, is not present in exponentially growing cells. Only when they are approaching the stationary phase, as detected by DNA flow cytometry, PDGF is able to induce an evident phosphorylation of the receptor.
*The antibodies were a kind gift of P.M.Cemoglio, Istituto di Istologia - Torino. I. Comoglio P.M., et al. (1984) EMBO J. 3, 483-489. 2. Frackelton A.R., et al. (1984) J. Biol. Chem. 259, 7909-7915. 3. Ek B., Heldin C.H. (1984) J. Biol. Chem. 259, 11145-11152. 4. Pledger W.J., et al. (1978) Prec. Natl. Acad. Sci. USA 75, 2839-2843. 5. Garrett J.S., et al. (1984) Prec. Natl. Acad. Sci. USA 81, 7466-7470. Zippel R. et al, (1986) Biochim. Biophys. Acta, in press. This work was supported by grants from CNR (Progetto Finalizzato Oncologia, n.84.00421. 44) and from Ministero Pubhlica Istruzione. tologia delle Membrane .
0309-1651/86/030164-01/$03.00/0
Gruppo di ricerca nazionale "Biologia e Pa-
~) 1986 Academic Press Inc. (London) Ltd.
Cell Biology International Reports, Vol. 10, No. 3, March 1986
AVAILABILITY OF PDGF RECEPTOR TO PDGF STIMULATED TYROSINE PHOSPHORYLATION IN MOUSE FIBROBLASTS IN DIFFERENT STATES OF GROWTH E. Sturani, R. Zippel, L. Toschi, L. Alberghina Dipartimento di Fisiologia e Biochimica Generali, UniversitY, Via Celoria, 26, Milano. Antibodies against phosphotyrosine (P-tyr)* (I) have allowed us to detect proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to PDGF. Western blotting of SDS PAGE fractionated proteins followed by decoration with P-tyr antibodies and 125I-protein A has been used (Zippel et al. Submitted for publication). The major tyrosine phosphorylated component appears to be the PDGF receptor in its tyrosine phosphorylated form as suggested by its molecular weight of 170 Kd (2~3), by the fact that it is detectable only in cell lines bearing the PDGF receptor (Swiss 3T~ NR6 and not in A431), and only in PDGF stimulated cells. At 37°C the phosphorylation is very rapid; the intensity of the 170 Kd band recognized by P-tyr antibodies is maximal after 5-10 min, afterwards it decreases (see also 2-3). The decrement seems not to be necessarily connected with internalization nor due to lysosomal degradation, since inhibitors of membrane internalization and of lysosomal proteolysis do not prevent the decrement of the band, which thus seems due to dephosphorylation rather than to proteolysis. This conclusion is supported by the finding that a shortage in ATP supply few min after PDGF stimulation makes the decrement more rapid, suggesting in addition that the overall phosphorylation is the net result of phosphorylation and dephosphorylation. At low temperature phosphorylation is slower but dephosphorylation (and internalization) de not take place. The PDGF concentration required for half maximal stimulation of receptor phosphorylation is about 0.5 nM (2) and is very close to that required to
induce competence acquisition during a two hours exposure to the factor. This concentration is 10-20 times higher than that required to allow entrance in S phase during a 24 hr exposure to PDGF and platelet poor plasma (4). Serum (10%-20%) stimulation, which allows S entrance, does not induce receptor phosphorylation during a 10 min to 6 hr exposure at 37°C, at least at the level of sen sitivity of our protocol. However stimulation with serum for long time (4 hrs) at 5°C allows the accumulation of the phospho rylated receptor. Thus presumably low PDGF concentrations which allow S entrance when left for a long time in contact with the cells, induce at 37°C the phosphorylation of a limited number of receptor molecules; since however the dephosphorylation is very rapid, the amount of the phosphorylated re ceptor remains at a very low steady state level, below the sensitivity of our method. 10% serum at 37°C causes downregulation of the receptor since in about 8 hr it removes from the surface of the quiescent cells the PDGF receptors as detectable by PDGF stimulated receptor phosphorylation (5). The same effect is also obtained treating the cells with PDGF and the larger is the PDGF concentration the higher becomes the rate of receptor removing. Moreover the PDGF r~ ceptor, as detected by PDGF stimulated receptor phosphorylation, is not present in exponentially growing cells. Only when they are approaching the stationary phase, as detected by DNA flow cytometry, PDGF is able to induce an evident phosphorylation of the receptor.
*The antibodies were a kind gift of P.M.Cemoglio, Istituto di Istologia - Torino. I. Comoglio P.M., et al. (1984) EMBO J. 3, 483-489. 2. Frackelton A.R., et al. (1984) J. Biol. Chem. 259, 7909-7915. 3. Ek B., Heldin C.H. (1984) J. Biol. Chem. 259, 11145-11152. 4. Pledger W.J., et al. (1978) Prec. Natl. Acad. Sci. USA 75, 2839-2843. 5. Garrett J.S., et al. (1984) Prec. Natl. Acad. Sci. USA 81, 7466-7470. Zippel R. et al, (1986) Biochim. Biophys. Acta, in press. This work was supported by grants from CNR (Progetto Finalizzato Oncologia, n.84.00421. 44) and from Ministero Pubhlica Istruzione. tologia delle Membrane .
0309-1651/86/030164-01/$03.00/0
Gruppo di ricerca nazionale "Biologia e Pa-
~) 1986 Academic Press Inc. (London) Ltd.