Identification of Candidate Substrates of SIK2 in vitro

Abstract

Purpose:: Our previous studies have shown the expression of SIK2 in all rat retinal cell layers. It has been proposed that SIK2 may take part in the crosstalk between RTK and GPCR pathways. PKA has been experimentally shown as the upstream SIK2 kinase. As PKA has been reported to modulate FGF/MAPK activation and FGF2 dependent transcription from FGF inducible response element (FIRE) requires cooperation with PKA pathways, we reasoned that SIK2 might be a potential regulator of FGF signal transduction in retina. The aim of this work is to explore potential SIK2 substrates that might take part in RTK pathways including FGF by testing the ability of SIK2 to phosphorylate selected RTK signal pathway elements.

Methods:: The motif search program at www.scansite.mit.edu/motifscanner was used to identify proteins that contain the SIK2 phosphorylation motif in SwissPort database. Among the identified potential substrates, RTK pathway members Gab1, Grb2 and A-Raf as well as the central regulator of FGF pathway, Frs2 were expressed in bacteria as GST fusion proteins, purified by affinity chromatography. The ability of SIK2 to phosphorylate these candidate substrates was determined by in vitro kinase assay.

Results:: The computational analysis revealed that Grb2, Gab1 and A-Raf have canonical SIK2 target motif. We have shown that of the three, Gab1 and A-Raf, that can be phosphorylated by SIK2 in vitro. Grb2 was not kinased by this enzyme. Likewise, Frs2 did not appear to be potential SIK2 substrates, despite of the high structure similarity of Frs2 to IRS1.

Conclusions:: We have shown that Gab1 and A-Raf are potential substrates of SIK2. These proteins are suggested to fine tune duration of ERK activation in a number of growth factor pathways, including FGF pathway, known to be important in the physiology and pathophysiology of retina. The duration of ERK activity suggested to be a determining factor in proliferation versus differentiation responses in some cell types. It is conceivable that the phosphorylation of Gab1 and A-Raf defined by SIK2 may regulate ERK activity, and subsequently, determine the cell fate. This study supports the fact that SIK2 may function at a key crosstalk position converging FGF and PKA-mediated pathways. In the light of these data, elucidation of the position and the role of SIK2 in retina may give some clues for the molecular basis of retinopathies.

Keywords: retina • signal transduction