The duration of phorbol-inducible ErbB2 tyrosine dephosphorylation parallels that of receptor endocytosis rather than threonine-686 phosphorylation: implications for the physiological role of protein kinase C in growth factor receptor signalling.
Tumour cell growth may be accelerated by protein kinase C (PKC) agonists such as phorbol esters and receptor tyrosine kinases, but receptor tyrosine kinases are in turn desensitized to growth factors by PKC agonists. To clarify this apparent PKC bifunctionality, we have used phosphoantibodies to determine the relationship between PKC-dependent phosphorylation events affecting the ErbB2 oncoprotein in G8/DHFR 3T3 cells. Neither the kinetics nor the extent of phorbol-induced juxtamembrane domain (Thr686) phosphorylation vary directly with C-terminal (Tyr1222) dephosphorylation, with Tyr1222 continuing to be dephosphorylated long after Thr686 phosphorylation has also declined. Platelet-derived growth factor (PDGF) mimics the short-term effects of phorbol on Thr686 and Tyr1222 phosphorylation, and confocal microscopy reveals that both of these PKC agonists induce rapid internalization of PKC-modified ErbB2. Phorbol causes sustained cytoplasmic accumulation of PKC-phosphorylated receptors, however, whereas PDGF triggers the appearance of this ErbB2 subset only briefly. Metabolic labelling and co-precipitation studies fail to implicate heterologous molecules in either the tyrosine dephosphorylation or internalization of PKC-modified ErbB2. Taken in the context of earlier juxtamembrane domain mutagenesis studies, these findings indicate that phorbol-activated PKC may desensitize growth factor receptors to extracellular ligands solely by triggering sustained receptor internalization. We submit that PKC-dependent juxtamembrane domain phosphorylation represents a physiological mechanism for shortening the duration and enhancing the specificity of growth factor signalling by promoting internalization of liganded and unliganded receptors, respectively.
Ouyang
X
X
Department of Metabolic Medicine, Imperial College School of Medicine, London, UK.
Gulliford
T
T
Epstein
R J
RJ
D016475:3T3 Cells; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D004705:Endocytosis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D018719:Erb-b2 Receptor Tyrosine Kinases; D017978:Receptors, Growth Factor; D013912:Threonine; D014443:Tyrosine; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse