Integrated modulation of phorbol ester-induced Raf activation in EL4 lymphoma cells

Abstract

The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that “PMA-sensitive” cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In “PMA-resistant” and “intermediate” EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation.

Curcumin Induces Apoptosis in EJ Bladder Cancer Cells via Modulating C-Myc and PI3K/Akt Signaling Pathway

“…We also found that blocking Akt by curcumin remarkably decreased the levels of phospho-Bad, phospho-GSK-3β, phospho-c-Raf and pro-caspase-9 in EJ cells, indicating an increasing activity of Bad, GSK-3β, c-Raf and caspase-9. Although c-Raf was activated by curcumin in EJ cells, it is unknown whether sufficient activation of c-Raf activates the Raf/Erk cascades, because c-Raf activity is also regulated by protein-protein interaction [ 60 ], further research will be required. Accordingly, the suppression of anti-apoptotic PI3K/Akt signaling pathway by curcumin is also an important mechanism of action in c-myc-induced EJ cell apoptosis.…”

Inhibition of RAF dimers: it takes two to tango

“…Dual phosphorylation of a specific SPKTP motif within BRAF and CRAF was shown to down-regulate ERK1/2 activation, [76], with phosphorylation of BRAF residues S750 and T753 impairing BRAF:CRAF dimerisation and reducing ERK1/2 activation [77]. Additionally, phosphorylation of S289, S296 and S301 of CRAF by ERK1/2 has also been shown to inactivate RAF signalling [78]. ERK1/2-dependent BRAF phosphorylation reduces heterodimer formation due to reduced affinity for RAS ( phosphorylation at S151) and direct disruption of BRAF:CRAF heterodimers ( pT401, pS750 and pT753) [79] As ERK1/2 feedback phosphorylation impairs RAF dimerisation it is effective at blocking wild-type RAF activity.…”

Cardiac cAMP: production, hydrolysis, modulation and detection

“…Thus, PKC-dependent phosphorylation of AC-C1 domain induces AC activation (Ebina et al, 1997 ). However, one must be cautious on the use of PMA as a specific AC-activator since PMA has also been reported to have actions on non-kinase proteins including chimaerins, RasGRP, and Unc-13/Munc-13 (Han and Meier, 2009 ; Kazanietz et al, 1995 ).…”