Activation of Cdk2 stimulates proteasome-dependent truncation of tyrosine phosphatase SHP-1 in human proliferating intestinal epithelial cells.
SHP-1 is expressed in the nuclei of intestinal epithelial cells (IECs). Increased SHP-1 expression and phosphatase activity coincide with cell cycle arrest and differentiation in these cells. Suspecting the tumor-suppressive properties of SHP-1, a yeast two-hybrid screen of an IEC cDNA library was conducted using the full-length SHP-1 as bait. Characterization of many positive clones revealed sequences identical to a segment of the Cdk2 cDNA sequence. Interaction between SHP-1 and Cdk2 was confirmed by co-immunoprecipitations whereby co-precipitated Cdk2 phosphorylated SHP-1 protein. Inhibition of Cdk2 (roscovitine) or proteasome (MG132) was associated with an enhanced nuclear punctuate distribution of SHP-1. Double labeling localization studies with signature proteins of subnuclear domains revealed a co-localization between the splicing factor SC35 and SHP-1 in bright nucleoplasmic foci. Using Western blot analyses with the anti-SHP-1 antibody recognizing the C terminus, a lower molecular mass species of 45 kDa was observed in addition to the full-length 64-65-kDa SHP-1 protein. Treatment with MG132 led to an increase in expression of the full-length SHP-1 protein while concomitantly leading to a decrease in the levels of the lower mass 45-kDa molecular species. Further Western blots revealed that the 45-kDa protein corresponds to the C-terminal portion of SHP-1 generated from proteasome activity. Mutational analysis of Tyr(208) and Ser(591) (a Cdk2 phosphorylation site) residues on SHP-1 abolished the expression of the amino-truncated 45-kDa SHP-1 protein. In conclusion, our results indicate that Cdk2-associated complexes, by targeting SHP-1 for proteolysis, counteract the ability of SHP-1 to block cell cycle progression of IECs.
Simoneau
Mélanie
M
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Boulanger
Jim
J
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Coulombe
Geneviève
G
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Renaud
Marc-André
MA
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Duchesne
Cathia
C
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Rivard
Nathalie
N
Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Universitéde Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Electronic address: Nathalie.Rivard@USherbrooke.ca.
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D007422:Intestines / Q000166:cytology*; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D020798:Two-Hybrid System Techniques
D004791:Enzyme Inhibitors; D007976:Leupeptins; D011687:Purines; D000077546:Roscovitine; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde