SIK3 involvement in FGF2 signaling in Müller cells

Abstract

FGF2 stimulation of Millier cells regulates several processes including cell proliferation and survival by triggering the activation RaslMAPK and PI3K1 Akt pathways. Data from our lab indicate that serine/threonine kinase SIK2 regulates FGF2 signaling pathway. Regarding the high level of sequence and functional similarity between SIK family proteins, we aimed to investigate the possibility of SIK3, which is also a serine/threonine kinase, being involved in FGF2 signaling in Millier cells. To understand if SIK3 is involved in FGF2 signaling pathway, we examined FGF2-dependent changes in the phosphorylation status and expression pattern of SIK3 in Muller cells. Our results showed that SIK3 undergoes a transient increase in total serine phosphorylation levels, peaking in 10 mins of FGF2 stimulation, whereas total threonine phosphorylation levels show a transient decrease in 5 mins of FGF2 stimulation and then recover back to its basal levels. We observed no tyrosine phosphorylation at any point of FGF2 stimulation. SIK3 protein expression, on the other hand, significantly increased in 10 mins of FGF2 stimulation and decreased back to initial levels. These results suggest that SIK3 may have a regulatory role in FGF2 signaling pathway in Muller cells. Based on the presence of ERK phosphorylation motif on SIK3 and paralel profiles of FGF2-dependent ERK and SIK2 activation, in the second part, we explored the possibility of ERK being an upstream regulatory kinase of SIK3 in the context of FGF2 signaling in Muller cells. Our data revealed that the inhibition of ERK activity prior to FGF2 treatment results in reduced serine phosphorylation of SIK3, whereas threonine phosphorylation was enhanced compared to the controls. These results are consistent with the hypothesis that ERK regulates SIK3 phosphorylation status in Muller cells in an FGF2-dependent manner. Whether ERK directly or indirectly regulates SIK3 phosphorylation remains to be elucidated.