Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells

Abstract

Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21(Cip1/WAF1)) of G(1)-S transition, consistent with the G(1) cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells.

Fig. 1.

Knockdown of intestinal cell kinase (ICK) impairs proliferation of intestinal epithelial cells. A and B: COLO 205 cells were infected with lentiviral human ICK short hairpin (sh) RNA-1, ICK shRNA-2, or the control shRNA. The protein levels of ICK were assessed by Western blotting (A). Infected COLO 205 cells, including cells floating in culture medium and cells removed from culture dishes by trypsinization, were stained with Trypan blue and counted with a hemocytometer. Total cell numbers are shown in B; means ± SD, n = 3, **P < 0.01. C and D: RIE and Caco-2 cells were infected with lentiviral rat ICK shRNA and human ICK shRNA-1, respectively, or the control shRNA. The protein levels of ICK were assessed by Western blotting (C). D: 4 days after infection, the viable cell number was determined in RIE and Caco-2 cells expressing ICK shRNA and expressed relative to the control shRNA (means ± SD, n = 3, **P < 0.01).

Fig. 2.

ICK deficiency triggers a significant cell cycle delay at G₁. After lentiviral shRNA infection, COLO 205 or RIE cells, including cells floating in culture medium, were harvested and stained with propidium iodide. The DNA content was determined by flow cytometry. The relative distribution of cells in the various phases of the cell cycle is shown.

Fig. 3.

ICK deficiency alters gene expression of cell cycle regulators at G₁. After lentiviral shRNA infection, COLO 205 cells were harvested as described in Fig. 2 and extracted for total cellular protein and mRNA. A: protein levels of cell cycle regulators in cells expressing ICK shRNA and the control shRNA were assessed by Western blotting. B: mRNA levels of cyclin D1 (CCND1) and c-Myc (c-myc) were determined by quantitative RT-PCR; means ± SD, n = 3, *P < 0.05.

Fig. 4.

Disrupting ICK function in intestinal epithelial cells impairs the expression and/or activity of key regulatory components of protein translation in the mTORC1 signaling pathway. After lentiviral shRNA infection, cells were harvested and extracted for total cellular protein. The protein levels of key components of the mTORC1 pathway and the classic MAPK pathway were assessed by Western blotting in COLO 205 cells (A) or Caco-2 cells (B) expressing either ICK shRNA or the control shRNA. mTOR, mammalian target of rapamycin; Raptor, regulatory associated protein of mTOR; S6K1, ribosomal protein S6 kinase (S6K1); eIF4E, eukaryotic initiation factor 4E; 4EBP1, eIF4E-binding protein 1.

Fig. 5.

ICK interacts with mTOR/Raptor in vivo and phosphorylates Raptor in vitro. A: glutathione S-transferase (GST)-ICK or GST was coexpressed with Flag-Raptor and/or AU1-mTOR in HEK293T cells. GST fusion proteins were pulled down from cell lysate and analyzed for association with Flag-Raptor or AU1-mTOR by Western blotting against the anti-Flag and anti-AU1 antibodies, respectively. B: Flag-mTOR or Flag-Raptor was expressed in HEK293T cells and isolated by anti-Flag immunoprecipitation prior to the in vitro kinase assay with His-ICK(1-296).

Fig. 6.

ICK deficiency in COLO 205 cells induces features of gene expression characteristic of colonic epithelial polarization and differentiation. After lentiviral shRNA infection, COLO 205 cells were harvested and extracted for total cellular protein. The protein levels of ICK, Cdx2, liver-intestine cadherin (LI-CDH), protocadherin LKC (PC-LKC), and E-cadherin were analyzed by Western blotting. Anti-β-catenin signal indicates equal loading of total proteins.

Fig. 7.

ICK deficiency in Caco-2 cells induces a significant increase in the mRNA level of sucrase isomaltase (SI), a marker for enterocytic differentiation. A: mRNA levels of ICK and SI were determined in Caco-2 cultures at different stages of confluence by quantitative RT-PCR; means ± SD, n = 3, *P < 0.05. B: mRNA levels of SI and Cdx2 in preconfluent replicating Caco-2 cells infected with either ICK shRNA or the control shRNA were determined by quantitative RT-PCR; means ± SD, n = 3, *P < 0.05.