Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.
We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.
Andreozzi
Francesco
F
Dipartimento di Biologia e Patologia Cellulare e Molecolare 'L Califano', Università degli Studi di Napoli 'Federico II', via S Pansini 5, 80131 Napoli, Italy.
Melillo
Rosa Marina
RM
Carlomagno
Francesca
F
Oriente
Francesco
F
Miele
Claudia
C
Fiory
Francesca
F
Santopietro
Stefania
S
Castellone
Maria Domenica
MD
Beguinot
Francesco
F
Santoro
Massimo
M
Formisano
Pietro
P
D016475:3T3 Cells; D000818:Animals; D004789:Enzyme Activation; D025461:Feedback, Physiological / Q000502:physiology; D066298:In Vitro Techniques; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*
D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C494999:Ret protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha