Lyuben M. Tsvetkov

p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27

Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27. Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27KIP1 through the ubiquitin E3 ligase SCFSKP2

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.

Chk2 activation and phosphorylation-dependent oligomerization

ABSTRACT The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.

Phosphorylation of Plk1 at S137 and T210 is inhibited in response to DNA damage.

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Plk1 is activated byphosphorylation at the G2/M phase boundary. Active Plk1 is involved in promotion ofmitotic entry through activation of Cdc25C, and through nuclear import of cyclin B1 thattogether activate Cdc2/cyclin B kinase. In earlier work, phosphopeptide mappingidentified several phosphorylation sites in Plk1. Mutational analysis pinpointed threonine210, which is located in the activation loop of the kinase domain, as the major activationsite of Plk1. In response to DNA damage, ATM/ATR-dependent checkpoint pathwaysinhibit Plk1 activity. Insensitivity of Plk1T210D, a constitutively active mutant, to DNAdamage-induced inhibition of Plk1 indicates that regulation of Plk1 phosphorylation is apotential target of DNA damage checkpoints. In the present paper, we report that in vivophosphorylation of Plk1 at serine 137 (S137) and threonine 210 (T210) occurs in mitosis.DNA damage prevents phosphorylation of Plk1 at both S137 and T210 in asynchronouscells but not in mitotic cells. Inhibitors of ATM/ATR and Chk1/Chk2 protein kinasesavert the inhibition of Plk1 phosphorylation in response to DNA damage. These datasuggest a participation of DNA damage checkpoints in regulation of the signalingpathways upstream of Plk1.

Promotion of S‐phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: Association with inhibition of p27 accumulation

The sensitive‐to‐apoptosis gene (SAG) was initially identified as a redox‐inducible, apoptosis‐protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S‐phase entry as determined by [3H]‐thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek‐1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin‐dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG‐induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG‐overexpressing cells, serum starvation induced 10‐ to 18‐fold accumulation of p27 in control Rhek‐1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5‐fold) in SAG‐infected cells. Inhibition of p27 accumulation was also observed in stably SAG‐overexpressing SY5Y cells. Significantly, SAG‐associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F‐box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG‐overexpressing cells grown under serum starvation. Thus, SAG‐induced growth with serum withdrawal appears to be associated with SAG‐mediated p27 degradation. Mol. Carcinog. 30:37–46, 2001.

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.

The Plk1 Polo box domain mediates a cell cycle and DNA damage regulated interaction with Chk2.

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Chk2 is a tumorsuppressor that participates in DNA damage checkpoint signaling cascades. Plk1phosphorylates, co-localizes with, and interacts with Chk2, suggesting interconnection ofDNA damage checkpoints and mitotic regulation. However, the function of theirassociation is unknown. Here, we show that the interaction between Chk2 and Plk1 is cellcycle-regulated, with a peak in mitosis. DNA damage in G2 and M phases but not in Sphase induces dissociation of Plk1 and Chk2. In vitro, the Plk1 PBD bindsphosphorylated Chk2, and mediates an interaction independent of other eukaryoticproteins. Additionally, a phosphopeptide encompassing phosphoT68 of Chk2 binds Plk1in a PBD-dependent manner, and stimulates Plk1 activity. These results identify potentialmechanisms for interaction and inter-regulation of these two protein kinases.

Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation

The cell cycle controls processes of DNA replication and segregation of replicated DNA into two daughter cells. These processes are coordinated by multiple signaling pathways, which employ many protein kinases. The members of the family of Polo‐like protein kinases are among these key cell cycle regulators. In response to DNA damage and inhibited DNA replication, DNA structure checkpoints delay cell cycle progression to provide cells with time for repair of damaged DNA and protect it from more severe damage. These effects are achieved by affecting key players of the basic cell cycle regulation of the cells with damaged DNA. This review is focused on the interplay between Chk2, a bona fide checkpoint protein kinase, and Polo‐like kinases. IUBMB Life, 56: 449‐456, 2004