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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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PKC activation inhibits cyclin E– and cyclin A–associated kinase activity in IEC-18 cells. (a) Histone H1 kinase activity in cyclin E and cyclin A immunoprecipitates was determined by in vitro kinase assays and SDS-PAGE autoradiography as described in Materials and Methods. Results shown are representative of three independent assays. n/a, not applicable. (b) Bar graphs depict average activity in three replicate samples as determined by densitometric analysis of autoradiograms. U, untreated.
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Figure 4: PKC activation inhibits cyclin E– and cyclin A–associated kinase activity in IEC-18 cells. (a) Histone H1 kinase activity in cyclin E and cyclin A immunoprecipitates was determined by in vitro kinase assays and SDS-PAGE autoradiography as described in Materials and Methods. Results shown are representative of three independent assays. n/a, not applicable. (b) Bar graphs depict average activity in three replicate samples as determined by densitometric analysis of autoradiograms. U, untreated.

Mentions: To examine the mechanisms underlying PKC-induced alterations in pocket protein phosphorylation, cyclin D–, E–, and A–cdk complexes were immunoprecipitated from control and PMA-treated cells, and their activity was determined in immune complex kinase assays (Fig. 4). Consistent with findings in other intestinal epithelial model systems (Tian and Quaroni 1999), cyclin D–associated kinase activity could not be detected in IEC-18 cells (data not shown), possibly as a result of the low levels of cyclin D expressed in these cells. On the other hand, kinase activity was readily detectable in cyclin E and cyclin A immunoprecipitates from untreated cells (Fig. 4), and the activity in both complexes was markedly inhibited after PMA treatment. Cyclin E–associated activity was decreased by 46% at 2 h and by 73% at 6 h. By 8 h, although still inhibited by 48%, cyclin E–cdk2 activity was beginning to rebound. Cyclin A–associated activity was decreased by 55% within 2 h of treatment, and was below the accurate detection limit of the assay at 6 and 8 h (see Fig. 5).


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)

PKC activation inhibits cyclin E– and cyclin A–associated kinase activity in IEC-18 cells. (a) Histone H1 kinase activity in cyclin E and cyclin A immunoprecipitates was determined by in vitro kinase assays and SDS-PAGE autoradiography as described in Materials and Methods. Results shown are representative of three independent assays. n/a, not applicable. (b) Bar graphs depict average activity in three replicate samples as determined by densitometric analysis of autoradiograms. U, untreated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169440&req=5

Figure 4: PKC activation inhibits cyclin E– and cyclin A–associated kinase activity in IEC-18 cells. (a) Histone H1 kinase activity in cyclin E and cyclin A immunoprecipitates was determined by in vitro kinase assays and SDS-PAGE autoradiography as described in Materials and Methods. Results shown are representative of three independent assays. n/a, not applicable. (b) Bar graphs depict average activity in three replicate samples as determined by densitometric analysis of autoradiograms. U, untreated.
Mentions: To examine the mechanisms underlying PKC-induced alterations in pocket protein phosphorylation, cyclin D–, E–, and A–cdk complexes were immunoprecipitated from control and PMA-treated cells, and their activity was determined in immune complex kinase assays (Fig. 4). Consistent with findings in other intestinal epithelial model systems (Tian and Quaroni 1999), cyclin D–associated kinase activity could not be detected in IEC-18 cells (data not shown), possibly as a result of the low levels of cyclin D expressed in these cells. On the other hand, kinase activity was readily detectable in cyclin E and cyclin A immunoprecipitates from untreated cells (Fig. 4), and the activity in both complexes was markedly inhibited after PMA treatment. Cyclin E–associated activity was decreased by 46% at 2 h and by 73% at 6 h. By 8 h, although still inhibited by 48%, cyclin E–cdk2 activity was beginning to rebound. Cyclin A–associated activity was decreased by 55% within 2 h of treatment, and was below the accurate detection limit of the assay at 6 and 8 h (see Fig. 5).

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