Nucleocytoplasmic Shuttling of the Retinoblastoma Tumor Suppressor Protein via Cdk Phosphorylation-dependent Nuclear Export

Abstract

The retinoblastoma (RB) tumor suppressor protein is a negative regulator of cell proliferation that is functionally inactivated in the majority of human tumors. Elevated Cdk activity via RB pathway mutations is observed in virtually every human cancer. Thus, Cdk inhibitors have tremendous promise as anticancer agents although detailed mechanistic knowledge of their effects on RB function is needed to harness their full potential. Here, we illustrate a novel function for Cdks in regulating the subcellular localization of RB. We present evidence of significant cytoplasmic mislocalization of ordinarily nuclear RB in cells harboring Cdk4 mutations. Our findings uncover a novel mechanism to circumvent RB-mediated growth suppression by altered nucleocytoplasmic trafficking via the Exportin1 pathway. Cytoplasmically mislocalized RB could be efficiently confined to the nucleus by inhibiting the Exportin1 pathway, reducing Cdk activity, or mutating the Cdk-dependent phosphorylation sites in RB that result in loss of RB-Exportin1 association. Thus RB-mediated tumor suppression can be subverted by phosphorylation-dependent enhancement of nuclear export. These results support the notion that tumor cells can modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.Mutations in cell cycle components that allow cells to bypass quiescence or cellular senescence pathways are important hallmarks of a cancer cell thereby making the cell cycle machinery an important target for anti-cancer therapeutic strategies (1, 2). Frequent alterations that lead to inactivation of RB tumor suppressor function include overexpression of cyclins and cyclindependent kinases (Cdks), 3 inactivation of Cdk inhibitors (CKIs) or loss of RB expression, all of which result in aberrant activation of Cdks (1). RB is active in its hypo or underphosphorylated state whereas sequential phosphorylation of the sixteen serine/threonine residues of RB by several Cdks leads to inactivation of RB function (3). Cdk-mediated phosphorylation alters association of RB with its myriad interacting proteins that regulate cell cycle progression and transformation potential (4). The association of RB with E2F transcription factors is the best characterized and it is believed that phosphorylation of RB on several Cdk phosphorylation sites results in the release of E2Fs and activation or repression of target E2F-dependent promoters (5).The normally nuclear localization of RB is facilitated by a bi-partite nuclear localization signal (NLS) in the C terminus (6). Underphosphorylated RB remains in the nucleus via association of its N terminus with nuclear matrix proteins (7). In contrast, G 1 /S phosphorylation results in decreased affinity of hyperphosphorylated RB for the nuclear compartment (8 -10). The mechanistic details that govern the fate of this hyperphosphorylated RB species and its physiological relevance in relation to RB tumor suppressor function have been obscure and are the subject of this report. We and others...

Aberrant nucleocytoplasmic localization of the retinoblastoma tumor suppressor protein in human cancer correlates with moderate/poor tumor differentiation

“…Treatment with flavopiridol resulted in reduced viability of PC-3 cells ( Figure 1a). Importantly, similar to our observations with flavopiridol-treated Cdk4 R24C cells (Jiao et al, 2006), flavopiridol treatment of PC-3 cells resulted in nuclear retention of RB (Figure 1b). A dose-dependent increase in nuclear retention of RB was observed with flavopiridol concentration between 0.1 and 0.5 mM (data not shown).…”

“…The karyopherin family of nuclear export receptors (Weis, 2003), notably Exportin1 (CRM1) (Stade et al, 1997), are involved in trafficking of diverse substrates across the nuclear membrane. Our recent studies uncovered an association between the RB and Exportin1 proteins in Cdk4 R/R cells, where Exportin1 preferentially recognizes and associates with RB phosphorylated on C-terminal residues and thereby mediates export of this phosphorylated RB species (Jiao et al, 2006). Consistent with the immunofluorescence experiments (Figure 1b), western blot analysis of nucleocytoplasmic fractions from PC-3 cells revealed nucleocytoplasmic localization of RB ( Figure 2a).…”

“…Transition through the G 1 /S boundary of the cell cycle leads to conversion of RB from a low salt resistant to low salt extractable hyperphosphorylated species (Mittnacht and Weinberg, 1991). Recently, we showed that RB is confined to the nucleus in normal human and mouse fibroblast cells during all stages of the cell cycle (Jiao et al, 2006). In contrast, nucleocytoplasmically localized RB was observed in fibroblasts harboring the oncogenic Cdk4 R24C mutation.…”

“…We demonstrated that the aberrant nucleocytoplasmic localization is facilitated by CDKdependent nuclear export of RB by Exportin1 (CRM1). Further, we showed that phosphorylation residues in the C-domain of RB are critical in determining its nucleocytoplasmic localization (Jiao et al, 2006).…”

“…The observation of cytoplasmic RB is intriguing since RB is widely believed to be a nuclear protein and nuclear retention is presumed to be important for its tumor suppressor function. Previously, we elucidated the occurrence of nucleocytoplasmically distributed RB in Cdk4 R/R mouse embryonic fibroblasts (Jiao et al, 2006). Here, we investigated the requirement for CDK activity in determining the nucleocytoplasmic distribution of RB by treating PC-3 cells with the potent CDK inhibitor flavopiridol (Sedlacek, 2001).…”