title: Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation.
abstract: The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src (Y527)  and Ras (V12)  activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.
authors.affiliation: Department of Cellular and Molecular Biology and Pathology, University of Naples Federico II, Naples, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Salzano
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Rusciano
authors.forename: Eleonora
authors.initials: E
authors.lastname: Russo
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bifulco
authors.forename: Loredana
authors.initials: L
authors.lastname: Postiglione
authors.forename: Mario
authors.initials: M
authors.lastname: Vitale
mesh_terms: D000818:Animals; D019556:COS Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D005353:Fibronectins; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D012694:Serine; D054462:p21-Activated Kinases; D018631:ras Proteins