Abstract
The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.
Highlights
The retinoblastoma tumor suppressor gene encodes a nuclear phosphoprotein1 that regulates the G1/S transition of the cell cycle
Dephosphorylation of pRB by mitotic cell extracts is sensitive to inhibitors of PP1 [16], and a high molecular weight form of PP1 has been isolated as a pRB phosphatase [18]. pRB can associate with the catalytic subunit of PP1 [19], and a constitutively active form of PP1 induces dephosphorylation of pRB and pRB-dependent cell cycle arrest [20]
The high concentration of okadaic acid (OA) required for complete inhibition of PP2A was similar to previous reports and was probably due to the 100-fold higher concentration required for membrane permeation rates equivalent to calyculin A (CL-A) [37]
Summary
PP2A-like phosphatases are more sensitive to okadaic acid than PP1, these results imply that the effects of this toxin in G1 may be due to suppression of PP2A. The differential sensitivities to these inhibitors has provided methods to identify and quantitate the levels of PP1 and PP2A in cell and tissue extracts [36] Several of these inhibitors including okadaic acid, calyculin A, and tautomycin are membranepermeable and potently inhibit phosphatase activity in intact cells. Due to their differential affinities for PP1 and PP2A and their distinct permeation properties, these three inhibitors can inhibit PP1 and PP2A in a highly selective manner [37]. We used these inhibitors to identify the roles and potential mechanisms of PP1 and PP2A in regulating pRB phosphorylation. PP2A, or a PP2A-like phosphatase, is crucial for activation or maintenance of G1 cyclindependent kinase activity, while PP1 directly dephosphorylates pRB
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