Relative role of upstream regulators of Akt, ERK and CREB in NCAM- and FGF2-mediated signalling.

Although a large number of signalling cascades are known to be activated downstream of NCAM, only little is known regarding the hierarchical relationship between the involved molecules in the individual cascades and the level of cross talk between the cascades. Here, we evaluated the requirement of putative upstream signalling cascades for the phosphorylation of the kinases extracellular signal-regulated kinase (ERK) and Akt and the transcription factor cyclic adenosine monophosphate (cAMP) response-element binding protein (CREB) following stimulation of NCAM in rat cerebellar granule neurons with an NCAM ligand, the C3d peptide. NCAM-mediated ERK phosphorylation depended on activation of the fibroblast growth factor receptor (FGFR), Src-family kinases, MEK (MAP and ERK kinase) and G(0)/G(i)-proteins, whereas NCAM-mediated CREB phosphorylation depended on the activity of Src-family kinases and MEK. NCAM-specific Akt phosphorylation depended on cyclic guanosine monophosphate (cGMP) and phosphatidylinositide 3-kinase (PI3K). All three phosphorylation events were independent of activation of the signalling molecules phospholipase C, protein kinase C, protein kinase A, and CamKII, which all have been demonstrated previously to be involved in NCAM signalling. For comparison, we also evaluated the role of upstream signalling cascades on fibroblast growth factor 2 (FGF2)-mediated phosphorylation of ERK, Akt, and CREB and found that FGF2 required the activity of both FGFR and Src-family kinases for phosphorylation of ERK, Akt, and CREB. MEK was required for phosphorylation of ERK and CREB, but not Akt, whereas G(0)/G(i)-proteins were necessary for phosphorylation of Akt and CREB, and cGMP was necessary for Akt phosphorylation. We thus demonstrate that even though NCAM and FGF2 have many signalling features in common, and even though both are known to activate FGFR, there are a number of differences in the intracellular signalling network activated by the NCAM ligand C3d and the FGFR ligand FGF2.