The Effect of SIN1 and Microtubules on Insulin Induced PKC ζ Activation.

BACKGROUND Protein kinase C zeta ζ) plays an important role in insulin induced glycometabolism and insulin receptor (IR) associated signaling pathways. The full activation of ζ depends on its translocation from cytosol to membrane and phosphorylation at Thr410. However, the mechanism of duanyu1531 ζ activation remains elusive. In this study, the effect of SIN1 and microtubules on insulin-induced duanyu1531 ζ activation was investigated. MATERIAL AND METHODS HepG2 cells were stimulated with insulin for co-immunoprecipitation (co-IP) assay. The immunocomplex was captured by using ζ, anti-SIN1 or anti-FLAG antibodies and was subjected to western blotting analysis for detecting duanyu1531 ζ, SIN1, and β-tubulin protein expression level. The cells were intervened by small interfering RNA (siRNA) that targeted exon regions of SIN1. Then the glucose uptake ratio after cells were stimulated by insulin was measured. The duanyu1531 ζ insulin receptor levels in the membranes were analyzed. Cells stained with anti-duanyu1531 ζ, anti-SIN1 antibodies and probed with molecular probes were observed by immunofluorescence confocal microscopy. RESULTS SIN1 interacted and co-located with duanyu1531 ζ by pleckstrin homology (PH) domain. Downregulation of SIN1 severely impaired duanyu1531 ζ translocation and phosphorylation induced by insulin. duanyu1531 ζ co-immunoprecipitated with β-tubulin at different intervals upon insulin stimulus, and the activation of duanyu1531 ζ was affected by paclitaxel and nocodazole. CONCLUSIONS duanyu1531 ζ translocated from cytosol to membrane depending on SIN1, which suggested that duanyu1531 ζ may be activated directly by PI3K and the reaction probably carried out on microtubules in HepG2 cells.

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