ERK, AKT and PKA as regulatory kinases of SIK2 in FGF signaling in muller cells

Abstract

FGF2 dependent retinal Müller cell proliferation is elicited mainly via activation of Ras/MAPK pathway. Phosphorylation/dephosphorylation of intermediates is one of the key mechanisms for the transient and rapid regulation of the pathway. Studies from our laboratory suggest serine/treonine kinase SIK2 is involved in this regulatory process. In this study we focused on defining potential upstream kinases of SIK2 in Müller cells in the context of FGF signaling. Presence of ERK phosphorylation motif on SIK2 led us to explore the possibiliy of SIK2 being a substrate of this dual kinase. Through the use of constitutively active ERK we showed phosphorylation of SIK2 in vitro. When the FGF dependent activation of ERK blocked by U0126 prior to growth factor stimulation, decrease in SIK2 activity and in its threonine phosphorylation were evident, while no change was observed in serine phosphorylation. Co-immunoprecipitation studies revealed that the two proteins interact in vivo in FGF dependent manner. ERK inhibition also led to a decrease in SIK2 protein levels in our cells. Based on these results we propose that ERK acts as an activatory kinase of SIK2, targeting threonine residues and modulate SIK2 stability. Co-immunoprecipitation of SIK2 with FGFR2 and Gab1 raises the possibility that ERK dependent SIK2 activation downregulates FGF signaling through the kinasing of these proteins.In the second part whether PKA and AKT can regulate SIK2 in Müller cells as reported in other cellular contexts was analyzed. Blocking of AKT activation or inhibition of PKA prior to FGF2 induction resulted in decrease of SIK2 serine phosphorylation and its activation. SIK2 serine phosphorylation increased significantly when PKA activator was used. Co-immunoprecipitation analysis confirmed PKA/SIK2 interaction in vivo. The results indicate that AKT and PKA act as inhibitory upstream kinases of SIK2 in FGF2 signaling.