Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1–dependent cellular transformation

Abstract

cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3␤ could inhibit nuclear import. Here, we report that GSK-3␤-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3␤, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division. During G1 phase, the D-type cyclins (D1, D2, D3) accumulate and assemble with either cyclin-dependent kinase 4 or 6 (CDK4 or CDK6) in response to mitogenic growth factors. The active cyclin D holoenzyme promotes G1 progression by inactivating the growth-suppressive properties of the retinoblastoma protein (Rb) through site-specific phosphorylation and by virtue of its ability to titrate CDK inhibitors such as p27Kip1 and p21Cip1 (Weinberg 1995;Sherr and Roberts 1999). Titration of p27Kip1 and p21 Cip1, in turn, facilitates activation of the cyclin E/CDK2 complex and subsequent entry and progression through the DNA synthetic (S) phase of the cell cycle. Although p27Kip1 and p21 Cip1 are effective inhibitors of cyclin E/CDK2 and cyclin A/CDK2 complexes, recent evidence demonstrates that they promote the assembly of cyclin D/CDK complexes (LaBaer et al. 1997) and are found in catalytically active cyclin D/CDK complexes in vivo (Cheng et al. 1999).Extracellular mitogens promote cellular proliferation via receptor-mediated signaling circuitry that ultimately converges on the cell cycle machine, and the D-type cyclins function as critical sensors of these signals. For example, receptor-dependent activation of Ras promotes the accumulation of active cyclin D1/CDK4 complexes via at least two pathways. First, activated Ras promotes transcription of the cyclin D1 gene through a kinase cascade involving Raf1, mitogen-activated protein kinase kinase (MEK), and the extracellular signal-regulated protein kinases (Albanese et al. 1995;Aktas et al. 1997;Lavoie et al. 1996;Winston et al. 1996;Kerkhoff and Rapp 1997;Weber et al. 1997). Assembly and activation of the cyclin D1/CDK4 holoenzyme are also augmented by this same pathway . Second, the rate of cyclin D1 proteasomal degradation is mediated by GSK-3␤-dependent phosphorylation of a single threonine residue (Thr-286) near the C terminus of cyclin D1 (Diehl et al. 1998). Mitogens inactivate GSK-3␤ via a pathway involving Ras, phosphatidylinositol 3-kinase, and protein kinase B/Akt (Rodriguez-Viciana et al.

Impact of differential cyclin D1 expression and localisation in prostate cancer

“…The fourth pattern was represented by those tumours devoid of cyclin D1 (2/36). These data are consistent with the in vitro observations that cyclin D1 can be stringently regulated as a function of subcellular localisation (Alt et al, 2000;Radu et al, 2003) and reveal that distinct localisation patterns are observed in prostate cancer. Previous studies have shown that cyclin D1 activity is strongly influenced by its subcellular localisation (Diehl and Sherr, 1997;Barr and Johnson, 2001;Lin and Gelman, 2002;Balasenthil et al, 2004;Alao et al, 2006;Sumrejkanchanakij et al, 2006).…”

Cyclin D1: polymorphism, aberrant splicing and cancer risk

“…By contrast, these same studies validated the prediction that cyclin D1b is constitutively nuclear in localization (Lu et al, 2003;Solomon et al, 2003). This was a critical observation, as constitutive nuclear localization of cyclin D1 (as achieved through mutation of T286) is known to promote oncogenic transformation (Alt et al, 2000). Strikingly, it was demonstrated that cyclin D1b harbors increased transforming capability (as compared to cyclin D1) (Lu et al, 2003;Solomon et al, 2003), thus indicating that cyclin D1b may serve as a more potent oncogene in human cancers.…”

“…Although highly invoked in the literature, there has not been a detailed analysis of the role for the PEST sequence in regulating cyclin D1 protein stability. In contrast, phosphorylation of the T286 residue has been shown to target the cyclin D1 protein for nuclear export and proteasomal degradation (Alt et al, 2000). Indeed, phosphorylationdependent destruction of cyclin D1 provides a critical barometer to prevent overexpression of cyclin D1 resulting from continuous mitogen-dependent cyclin D1 transcription.…”

Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression

“…The level of cyclin D1 is tightly controlled for the normal progression of cell cycle and its deregulation is linked to the development of cancer (Bartkova et al, 1994a, b;BaniHani et al, 2000). Overexpression of wild-type cyclin D1 is shown to be insufficient in inducing transformation of cells, because nuclear export and subsequent cytoplasmic proteolysis reduce the oncogenic capability of the protein (Quelle et al, 1993;Alt et al, 2000). In contrast, overexpression of cyclin D1-T286A results in tumorigenesis in transgenic mice (Alt et al, 2000;Gladden and Diehl, 2005;Benzeno et al, 2006;Lin et al, 2008).…”

“…Activated AKT phosphorylates the serine9 residue of glycogen synthase kinase-3b (GSK3b) to inactivate its kinase activity on the threonine286 residue of cyclin D1. This later blocks the nuclear export and the cytoplasmic cyclin D1 and undergoes proteasomal degradation (Alt et al, 2000). Thus cyclin D1 accumulates in the nucleus as a result of the phosphatidylinositol-3-OH kinase-and AKT-mediated inactivation of GSK3b.…”