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28 July 2017 : Laboratory Research  

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

Xiang Yingying1ABCEF, Wang Caijuan1ACDE, Yue Yenan1D, Tang Yuqin1D, Cai Xueqin1D, Wu Zhongming1ADEFG*

DOI: 10.12659/MSM.905555

Med Sci Monit 2017; 23:3666-3672

Abstract

BACKGROUND: Protein kinase C zeta (PKC ζ) plays an important role in insulin induced glycometabolism and insulin receptor (IR) associated signaling pathways. The full activation of PKC ζ depends on its translocation from cytosol to membrane and phosphorylation at Thr410. However, the mechanism of PKC ζ activation remains elusive. In this study, the effect of SIN1 and microtubules on insulin-induced PKC 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 anti-PKC ζ, anti-SIN1 or anti-FLAG antibodies and was subjected to western blotting analysis for detecting PKC 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 PKC ζ insulin receptor levels in the membranes were analyzed. Cells stained with anti-PKC ζ, anti-SIN1 antibodies and probed with molecular probes were observed by immunofluorescence confocal microscopy.

RESULTS: SIN1 interacted and co-located with PKC ζ by pleckstrin homology (PH) domain. Downregulation of SIN1 severely impaired PKC ζ translocation and phosphorylation induced by insulin. PKC ζ co-immunoprecipitated with β-tubulin at different intervals upon insulin stimulus, and the activation of PKC ζ was affected by paclitaxel and nocodazole.

CONCLUSIONS: PKC ζ translocated from cytosol to membrane depending on SIN1, which suggested that PKC ζ may be activated directly by PI3K and the reaction probably carried out on microtubules in HepG2 cells.

Keywords: Insulin, Microtubules, Protein Kinase C

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750