The regulation of steroid receptor coactivator-3 (SRC-3) activity by ERK3-MK5 signal pathway. A study in lung cancer cells

Abstract

Background: Protein kinases and phosphatases persistently regulate various signal pathways that mediate many cellular processes. The mitogen activated protein kinases (MAPKs) are key components in the transduction of extracellular stimuli to biological responses. The dysregulation of MAPKs activity promotes the occurrence of diverse diseases, including cancer, making MAPKs signaling pathways attractive targets for developing new potential drugs. The extracellular signal-regulated kinase 3 (ERK3) is a unique atypical MAPK when it comes to regulation and functions. Much less is revealed about ERK3 pathophysiological functions in cancer pathogenesis. The MAPK-activated protein Kinase 5 (MK5) has lately been identified as the first downstream target for ERK3. A recent study has found that ERK3 can regulate the activity of the oncogenic steroid receptor coactivator 3 (SRC-3), which in turn promotes lung cancer cells migration and invasiveness.

Aim: In this study, we investigate the identity of the phosphor-donor of SRC-3 at Ser857, and examine the effect MK5, ERK3, and SRC-3 have on MMP2, MMP9, and MMP10 gene expressions, and on lung cancer cell proliferation and migration.

Methods: Subcloning, Western blot, qRT-PCR, luciferase assay, protein purification, in vitro kinase assay, colonogeneic assay, and scratch assay.

Results: MK5 seems to phosphorylate SRC-3 at Ser857 in vitro. SRC-3 and MK5 overexpression and co-overexpression increase MMP2 and MMP10 promoter activities. MK5, SRC-3, and ERK3 overexpression enhances MMP9 promoter activity. The siRNA-mediated knockdown of MK5 and ERK3 results in reduced MMP2, MMP9, and MMP10 promoter activity in A549 cells. Endogenous MMP2 and MMP9 mRNA levels significantly decrease by expressing shRNAs targeting MK5 and ERK3 in H1299 lung cancer cells.

Conclusion: MK5 phosphorylates SRC-3 at the S857 in vitro. MMP2, MMP9, and MMP10 promoter activity and MMP2 and MMP9 mRNA expression diminishes by RNAi-facilitated knockdown (either by siRNA or shRNA) of ERK3 and MK5 in lung cancer cells.