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Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD - J. Cell Biol. (2000)

Bottom Line: PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes.These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6.Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. jennifer.black@roswellpark.org

ABSTRACT
Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

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Altered expression and phosphorylation of the pocket proteins p107, pRb, and p130 after PKC activation in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA for the indicated times (U, untreated), and expression and phosphorylation state of pocket proteins were determined by Western blot analysis. Altered phosphorylation of these molecules is reflected in characteristic changes in their migration patterns on SDS gels. p130 is detected as forms 1, 2, and 3; the accumulation of forms 1/2 after PMA treatment is a hallmark of cell cycle withdrawal into G0. Data are representative of at least three independent experiments.
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Figure 2: Altered expression and phosphorylation of the pocket proteins p107, pRb, and p130 after PKC activation in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA for the indicated times (U, untreated), and expression and phosphorylation state of pocket proteins were determined by Western blot analysis. Altered phosphorylation of these molecules is reflected in characteristic changes in their migration patterns on SDS gels. p130 is detected as forms 1, 2, and 3; the accumulation of forms 1/2 after PMA treatment is a hallmark of cell cycle withdrawal into G0. Data are representative of at least three independent experiments.

Mentions: To investigate the role of pocket proteins in PKC-mediated cell cycle arrest in IEC-18 cells, the effects of PKC activation on the expression and phosphorylation state of these molecules were determined using Western blot analysis. This analysis was based on well-documented evidence that alterations in phosphorylation state of pocket proteins are reflected in characteristic changes in their migration patterns on SDS-PAGE gels (Ludlow et al. 1990; DeCaprio et al. 1992; Whyte and Eisenman 1992; Garriga et al. 1998; Smith et al. 1998; Thomas et al. 1998). As shown in Fig. 2, proliferating (untreated) IEC-18 cells express p107, pRb, and small amounts of p130; p107 and pRb are detected primarily in their hyperphosphorylated (i.e., slower-migrating), growth-permissive forms. Treatment with 100 nM PMA resulted in decreased expression and hypophosphorylation (i.e., appearance of characteristic faster-migrating forms) of p107 and pRb, and a marked accumulation of the faster-migrating forms 1/2 of p130 (which is characteristic of cell cycle exit into G0; see Mayol et al. 1995). These PMA-induced alterations were transient, reversing by 12–16 h after addition of agonist, coincident with release from cell cycle arrest as a consequence of PKC downregulation (see Fig. 1d and Fig. e).


Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD - J. Cell Biol. (2000)

Altered expression and phosphorylation of the pocket proteins p107, pRb, and p130 after PKC activation in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA for the indicated times (U, untreated), and expression and phosphorylation state of pocket proteins were determined by Western blot analysis. Altered phosphorylation of these molecules is reflected in characteristic changes in their migration patterns on SDS gels. p130 is detected as forms 1, 2, and 3; the accumulation of forms 1/2 after PMA treatment is a hallmark of cell cycle withdrawal into G0. Data are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169440&req=5

Figure 2: Altered expression and phosphorylation of the pocket proteins p107, pRb, and p130 after PKC activation in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA for the indicated times (U, untreated), and expression and phosphorylation state of pocket proteins were determined by Western blot analysis. Altered phosphorylation of these molecules is reflected in characteristic changes in their migration patterns on SDS gels. p130 is detected as forms 1, 2, and 3; the accumulation of forms 1/2 after PMA treatment is a hallmark of cell cycle withdrawal into G0. Data are representative of at least three independent experiments.
Mentions: To investigate the role of pocket proteins in PKC-mediated cell cycle arrest in IEC-18 cells, the effects of PKC activation on the expression and phosphorylation state of these molecules were determined using Western blot analysis. This analysis was based on well-documented evidence that alterations in phosphorylation state of pocket proteins are reflected in characteristic changes in their migration patterns on SDS-PAGE gels (Ludlow et al. 1990; DeCaprio et al. 1992; Whyte and Eisenman 1992; Garriga et al. 1998; Smith et al. 1998; Thomas et al. 1998). As shown in Fig. 2, proliferating (untreated) IEC-18 cells express p107, pRb, and small amounts of p130; p107 and pRb are detected primarily in their hyperphosphorylated (i.e., slower-migrating), growth-permissive forms. Treatment with 100 nM PMA resulted in decreased expression and hypophosphorylation (i.e., appearance of characteristic faster-migrating forms) of p107 and pRb, and a marked accumulation of the faster-migrating forms 1/2 of p130 (which is characteristic of cell cycle exit into G0; see Mayol et al. 1995). These PMA-induced alterations were transient, reversing by 12–16 h after addition of agonist, coincident with release from cell cycle arrest as a consequence of PKC downregulation (see Fig. 1d and Fig. e).

Bottom Line: PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes.These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6.Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. jennifer.black@roswellpark.org

ABSTRACT
Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Show MeSH