TY - JOUR
T1 - Protein phosphatase 2A associates with Rb2/p130 and mediates retinoic acid-induced growth suppression of ovarian carcinoma cells.
AU - Vuocolo, Scott
AU - Purev, Enkhtsetseg
AU - Zhang, Dongmei
AU - Bartek, Jiri
AU - Hansen, Klaus
AU - Soprano, Dianne Robert
AU - Soprano, Kenneth J
N1 - Keywords: Cell Division; Cell Line, Tumor; Endopeptidases; Female; Humans; Ovarian Neoplasms; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Binding; Protein Phosphatase 2; Proteins; Retinoblastoma-Like Protein p130; Tretinoin; Ubiquitins; Up-Regulation
PY - 2003
Y1 - 2003
N2 - Levels of Rb2/p130 protein are increased 5-10-fold following all-trans-retinoic acid (ATRA) treatment of the retinoid-sensitive ovarian adenocarcinoma cell line CAOV3, but not the retinoid-resistant adenocarcinoma cell line SKOV3. We found that this increase in Rb2/p130 protein levels in ATRA-treated CAOV3 cells was the result of an increased protein stability. Moreover, Rb2/p130 exhibited a decreased ubiquitination following ATRA treatment. Because phosphorylation frequently mediates ubiquitination of proteins, we examined the serine/threonine phosphatase activity in our CAOV3 cells following ATRA treatment. A significant increase in Ser/Thr phosphatase activity was found, which correlated with a rise in the level of protein phosphatase 2A (PP2A) catalytic subunit-alpha. In addition, co-immunoprecipitation and glutathione S-transferase pull-down studies demonstrated that PP2A and Rb2/p130 associate. We have made use of a battery of Rb2/p130 mutants to determine the sites dephosphorylated in response to ATRA treatment of CAOV3 cells. Obligate CDK4 phosphorylation sites seemed most important to the stability of the protein and are among the candidate sites that are dephosphorylated by PP2A following ATRA treatment. Finally, using both small interfering RNA specific to the catalytic subunit of PP2A and a variant of the SKOV3 cell line that overexpresses PP2A, we have shown that modulation of PP2A protein levels correlates with the ability of ATRA to inhibit growth of ovarian carcinoma cells. Our data suggest that ATRA mediates growth inhibition by stabilizing Rb2/p130 via a mechanism that involves induction of PP2A, an enzyme that can potentially dephosphorylate Rb2/p130, thereby protecting it from degradation by the proteasome.
AB - Levels of Rb2/p130 protein are increased 5-10-fold following all-trans-retinoic acid (ATRA) treatment of the retinoid-sensitive ovarian adenocarcinoma cell line CAOV3, but not the retinoid-resistant adenocarcinoma cell line SKOV3. We found that this increase in Rb2/p130 protein levels in ATRA-treated CAOV3 cells was the result of an increased protein stability. Moreover, Rb2/p130 exhibited a decreased ubiquitination following ATRA treatment. Because phosphorylation frequently mediates ubiquitination of proteins, we examined the serine/threonine phosphatase activity in our CAOV3 cells following ATRA treatment. A significant increase in Ser/Thr phosphatase activity was found, which correlated with a rise in the level of protein phosphatase 2A (PP2A) catalytic subunit-alpha. In addition, co-immunoprecipitation and glutathione S-transferase pull-down studies demonstrated that PP2A and Rb2/p130 associate. We have made use of a battery of Rb2/p130 mutants to determine the sites dephosphorylated in response to ATRA treatment of CAOV3 cells. Obligate CDK4 phosphorylation sites seemed most important to the stability of the protein and are among the candidate sites that are dephosphorylated by PP2A following ATRA treatment. Finally, using both small interfering RNA specific to the catalytic subunit of PP2A and a variant of the SKOV3 cell line that overexpresses PP2A, we have shown that modulation of PP2A protein levels correlates with the ability of ATRA to inhibit growth of ovarian carcinoma cells. Our data suggest that ATRA mediates growth inhibition by stabilizing Rb2/p130 via a mechanism that involves induction of PP2A, an enzyme that can potentially dephosphorylate Rb2/p130, thereby protecting it from degradation by the proteasome.
U2 - 10.1074/jbc.M302715200
DO - 10.1074/jbc.M302715200
M3 - Journal article
C2 - 12915404
SN - 0021-9258
VL - 278
SP - 41881
EP - 41889
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -