Login to Access Video or Poster Abstract: MP60-04
Sources of Funding: This project was supported by the national Natural Science Foundation of China (No.81572529).

Introduction

T-LAK cell-originated protein kinase (TOPK), a serine/threonine protein kinase, was reported highly expressed in a variety of human tumors, and associated with poor prognosis of many kinds of human malignancies, but its activation mechanism is still uncovered. It is well known there is a bidirectional signals transduced between TOPK and ERK2, and ERK2 could phosphorylate TOPK at Thr9. The objective of the present study is to explore whether there is other site on TOPK that can be phosphorylated by ERK2, the function of the phosphorylation site was also be investigated.

Methods

Potential phosphorylated serine/threonine sites of TOPK were predicted by NetPhos 2.0 software program. Peptide mapping assay was used to testify the phosphorylation site. Anti-phospho-TOPK at S32 was prepared, the endogenous phosphorylation of TOPK at S32 was detected in renal cancer cells and tissues of renal cancer patients. In order to investigate the function of S32 site, S32 mutated TOPK (S32A) was transient transfected into 293T cells and stably transfected into JB6 or Caki-1 cells, the in vitro kinase assay was performed, the anchorage-independent growth ability and tumorigenesis of TOPK-S32A cells were compared. The ERK2 was knocked down from the TOPK highly expressed renal cancer cells, the level of phosphorylation TOPK (S32) and other downstream genes were detected.

Results

We found that ERK2 directly bound with and phosphorylated TOPK at S32 in vitro. The phosphorylation was inhibited in cells expressing low levels of ERK2 or the cells that ERK2 was knocked down. When the S32 mutated TOPK was stably transfected into JB6, the anchorage-independent growth ability and tumorigenesis of the cells were suppressed compared with those of wild type TOPK (TOPK-WT) ex vivo and in vivo. The phosphorylation level of TOPK substrate, Histone H3 at Ser10 was also decreased dramatically ex vivo or in vivo. The phosphorylation level of TOPK was higher in high stage tissues of renal cancer than those in low stage ones.

Conclusions

Taken together, the phosphorylation of TOPK at S32 by ERK2 increases the activity of TOPK, and promotes the tumorigenesis of renal cancer. It may provide opportunities for TOPK based prognosis and targeted therapy for renal cancer patients.

Funding

This project was supported by the national Natural Science Foundation of China (No.81572529).