A second RING to destroy p27Kip1

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N E W S A N D V I E W S

Regulated degradation of proteins by the ubiq-uitin–proteasome system is a crucial mecha-nism that drives progression through the cell cycle. Ubiquitination involves an enzy-matic cascade that results in ubiquitin being covalently attached to a lysine residue on the substrate. Substrate specificity is imparted by E3 ubiquitin ligases that facilitate transfer of ubiquitin from an E2 conjugating enzyme to the substrate.

The cyclin-dependent kinase (CDK) inhibi-tor p27, a key cell-cycle regulator, becomes increasingly unstable as cells approach S phase. Degradation of p27 in S phase requires its phos-phorylation on a threonine residue (Thr 187). CDK2, which is inhibited by p27 in G1 phase, can phosphorylate this residue and thereby generates a binding site for the E3 ligase SCF-Skp2 (Skp1–Cul1–F-box protein). SCF–Skp2 polyubiquitinates p27 and targets it for deg-radation by the 26S proteasome1 (Fig. 1). The SCFSkp2 pathway for p27 degradation requires the activity of CDKs that are themselves inhib-ited by p27, suggesting the existence of a posi-tive feedback loop that ensures inhibitor levels remain low after CDK activation and thus contributes to the irreversibility of the G1–S transition. The decision to activate CDKs and to trigger this feedback loop must require addi-tional pathways to regulate p27 or CDKs.

Transcriptional and translational control, sequestration in cyclin D complexes and locali-zation all regulate p27 in G1 phase (refs 2–5). In addition to these mechanisms, two lines of evidence suggest that alternative proteolytic pathways control p27 stability in G1. One such pathway was identified in a mutant mouse where the endogenous p27 gene was exchanged for one encoding a p27-T187A mutant6. Surprisingly, this mutant p27 — which can-not be ubiquitinated by the T187-dependent SCF–Skp2 pathway — was still unstable after

serum stimulation. As cells progressed into S phase, the wild-type protein was further desta-bilized, whereas the T187A mutant regained stability. This suggested a novel proteolytic pathway for p27 that functions in G1 phase but is inactive during S phase. It does not require Thr 187 phosphorylation, but was not detected in cells lacking Skp2 (refs 6,7).

Evidence for a Skp2-independent pathway of p27 degradation came from an earlier study by Nakayama and colleagues8. Analysis of p27 ubiquitination in lymphocytes from Skp2-knockout mice uncovered a cytosolic activity that required neither T187 phosphorylation nor Skp2 and is present in G0 and S–G2 extracts8.

Both of the studies discussed above6,8 iden-tified a T187 phosphorylation-independent pathway of p27 degradation in G1 phase; but they differ in their requirement for Skp2 (refs 6–9). In the paper discussed next, the Skp2-independent pathway has been defined at the molecular level. It remains to be determined whether alternative independent pathways for p27 degradation exist in G1, and whether they function in different tissues or cell states.

Following up on their initial observations, on page 1229 of this issue Nakayama and col-leagues9 report the identification of a novel cytosolic ubiquitin ligase for p27 that seems to fulfil the requirements of their previously

A second RING to destroy p27Kip1

Ludger Hengst

The cell cycle regulator p27Kip1 must be degraded to permit cell division. Degradation is moderate in G1 phase, but is enhanced in S-phase. Now, a novel ubiquitin ligase that can ubiquitinate p27 leading to its proteolysis after mitogen stimulation has been identified.

Ludger Hengst is at the Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.e-mail: [email protected]

UbUb

UbUb

UbUb

UbUb KPC2

KPC1

Ubc4/H5A

p27

p27 p27 p27

Ubc3

Roc1Cu11

Skp1

Skp2

Cks1

P

G0 G1 S G2

Cytoplasm

Nucleus

Figure 1 Consecutive ubiquitination pathways regulate p27 proteolysis before and after the G1–S transition. These pathways involve two multi-subunit RING-finger E3 ubiquitin ligases, KPC and SCFSkp2 (green, RING finger subunits Kpc1 and Roc1) that cooperate with different E2 enzymes to ubiquitinate p27. In G1 phase, the KPC pathway requires export of p27 to the cytoplasm but it is phosphorylation-independent. In S phase, the SCFSkp2 pathway is suggested to function within the nucleus and is dependent on phosphorylation of p27 on Thr 187.

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described ubiquitination activity. Using a clas-sic protein purification approach combined with an in vitro p27 ubiquitination assay, they purified a protein complex with E3 ligase activ-ity from rabbit reticulocyte lysate. Peptide sequencing led to the identification of the novel ubiquitin ligase which they call KPC (Kip ubiq-uitination-promoting complex), composed of two subunits: KPC1 (with a relative molecular mass (Mr) of 140,000) and KPC2 (with an Mr of 50,000). KPC1 contains an amino-termi-nal SPRY domain of yet unknown function, and a carboxy-terminal RING finger — a motif required for E3 activity that is present in various E3 ubiquitin ligases, including the Roc1 subunit of the SCF complex (Fig. 1). Recombinant KPC1 ubiquitinates p27 in vitro and this activity depends on the RING motif as its deletion abolishes p27 ubiquitination. Out of seven E2 enzymes tested, two — Ubc4 and UbcH5A — cooperated with KPC in mediating p27 ubiquitination (Fig. 1).

The second subunit, KPC2, is a protein previously designated GBDR1 (glioblastoma cell differentiation related gene 1). GBDR1 mRNA was identified as a protein kinase Cα-upregulated transcript associated with breast tumour aggressiveness10, and is expressed in all human tissues investigated11. With an N-terminal ubiquitin-like (UBL) domain and two C-terminal ubiquitin-associated (UBA) domains, KPC2 shares structural similarities with Rad23. Rad23 and related multi-ubiq-uitin-chain-binding proteins can function in recognizing specific polyubiquitinated pro-teins and recruiting them to the proteasome. For example, yeast Rad23 recruits the polyu-biquitinated yeast CDK inhibitor Sic1 to the proteasome12. It is therefore tempting to specu-late that KPC2 might contribute to targeting polyubiquitinated p27 to the 26S proteasome in a similar way.

Somewhat unexpectedly, recombinant KPC2 reduced the ubiquitin ligase activity of KPC1. This finding is consistent with the idea that KPC2 functions in a Rad23-like manner, as Rad23 can also inhibit multi-ubiquitin-chain assembly in vitro13. As KPC activity was found only in fractions containing both subunits, full activity might require a modification of the complex or another component that was missed. Comparison of the specific activ-ity of purified compared with recombinant complexes may help address this question. Immunoprecipitation experiments with anti-KPC2 antibodies from extracts derived from

primary cultures of human microvascular endothelial cells (HMVEC) show that KPC co-precipitates with an unknown protein with an Mr of about 70K11. It is not clear whether this protein is ubiquitously expressed and whether it is a stoichiometric cofactor, but it will be inter-esting to test its effect on KPC ligase activity. KPC directly binds to p27 in vivo and in vitro. Whether p27-interacting proteins inhibit KPC binding or regulate KPC activity also remains to be determined.

How important is KPC for p27 regulation and cell-cycle progression? To address this, Nakayama and colleagues first overexpressed the KPC complex in serum-starved cells and followed the decline of p27 after stimulation. KPC-overexpressing cells had similar p27 protein levels to control cells; however, KPC overexpression accelerated the decline in p27 levels in G1 phase. If KPC1 lacking its RING-finger domain was coexpressed with KPC2, p27 decline was strongly delayed. In a second experi-ment, Nakayama and colleagues depleted KPC1 using RNA interference. As seen with the domi-nant-negative RING-finger deletion mutant, knockdown of KPC1 impaired p27 decline after serum stimulation. Consistent with their initial hypothesis that KPC1 degrades p27 selectively in G1 phase, the authors found that in pulse-chase experiments depletion of KPC1 stabilized p27 in G1- but not in S-phase cells. Analysis of p27 abundance and stability in Skp2–/– mouse embryonic fibroblasts (MEFs) revealed that KPC depletion in these cells strongly diminished the G1-specific decline of p27, almost completely abolishing its decline after serum stimulation. Taken together, these data demonstrate that KPC and SCF–Skp2 function independently to destabilize p27 in succession.

Some food for thought comes, however, from the puzzling observation that KPC depletion fails to influence progression from G0 to S phase, despite increased p27 levels. Does this mean that KPC is not required for the progression from G0 to S phase or that a second mechanism complements the depletion of KPC? For example, cytoplasmic sequestra-tion of p27 might lead to activation of nuclear CDKs, despite the presence of elevated p27 (ref. 5). Depletion of KPC1 in Skp2–/– MEFs delayed progression from G0 into S phase, which occurred (in reduced cell numbers) despite the unvaried high p27 level. A more detailed analysis of CDK activity, p27 localiza-tion or modifications may identify mechanisms that enable G1 progression in these cells. As

Skp2 has — and KPC may have — additional substrates besides p27, MEFs lacking p27 in addition to Skp2 were also analysed. Additional deletion of p27 was recently shown to restore most of the cellular phenotypes of Skp2-knock-out animals, confirming p27 as a central sub-strate for Skp2 (refs 7,14). Depletion of KPC in these cells did not alter progression towards S phase, indicating that p27 remains the key substrate in KPC1-/Skp2-deficient cells.

Finally, the authors addressed the question of how KPC accomplishes cell-cycle regulated p27 degradation. One obvious level of regula-tion is defined by the localization of KPC and p27. In most cells, p27 is predominantly local-ized to the nucleus, whereas KPC is localized in the cytoplasm. However, there is no evidence for regulation of KPC activity or localization. As p27 shuttles between the nucleus and the cytoplasm15, the degree of p27 export and import may directly determine whether it is ubiquitinated by KPC. To test this idea, the authors first used leptomycin B, an inhibitor of CRM1-dependent nuclear export. Indeed, leptomycin B impaired regulation of p27 by KPC. However, the data do not support a sim-ple model in which p27 would become consti-tutively unstable if exported to the cytoplasm. For example, p27 with a mutant nuclear locali-zation signal (p27-NLS) localizes to the cyto-plasm but it exhibits enhanced stability that is unaffected by depletion of KPC. One possible explanation for this is that degradation of p27 by the KPC pathway may require some preced-ing modification of p27 in the nucleus. This modification may be cell-cycle regulated, thus restricting KPC-dependent proteolysis of p27 to a window in G1 phase, even if p27 shuttles throughout the cell cycle. This model may also explain the accumulation of cytoplasmic p27 observed after AKT/PKB activation or in some tumours4,5. Of course, cytoplasmic p27 accu-mulation in tumours may also reflect impaired degradation by the KPC pathway. As Skp2 functions as an oncogene1,4,5, it will be inter-esting to determine whether KPC contributes to human malignancies.

In summary, the study by Nakayama and colleagues provides not only a fascinating new mechanism by which p27 can be sequentially degraded, but also evidence that p27 regulation is far from fully understood.

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2. Medema, R. H., Kops, G. J., Bos, J. L. & Burgering, B. M. Nature 404, 782–787 (2000).




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4. Slingerland, J. & Pagano, M. J. Cell. Physiol. 183, 10–17 (2000).

5. Viglietto, G., Motti, M. L. & Fusco, A. Cell Cycle 1, 394–400 (2002).

6. Malek, N. P. et al. Nature 413, 323–327 (2001).7. Kossatz, U. et al. Genes Dev. 18, 2602–2607 (2004).

8. Hara, T., Kamura, T., Nakayama, K., Oshikawa, K. & Hatakeyama, S. J. Biol. Chem. 276, 48937–48943 (2001).

9. Kamura, T. et al. Nature Cell Biol. 6, 1229–1235 (2004).

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Genomics 65, 243–252 (2000).12. Verma, R., Oania, R., Graumann, J. & Deshaies, R. J.

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Invasive growth: A two-way street for semaphorin signallingPaolo M. Comoglio, Luca Tamagnone and Silvia Giordano

Growing evidence suggests that semaphorins — known to provide directional cues during axon guidance — also provide regulatory signals for cell migration during tissue morphogenesis. During heart development, it is crucial that semaphorins can signal bidirectionally, functioning as both a ligand and a receptor. Through these distinct signalling pathways, semaphorins can provide both ‘stop’ and ‘go’ signals for cell motility and invasive growth.

With few notable exceptions, the biological response is unidirectional for most signal–receptor systems and is transduced by the receptor. Indeed, in the case of semaphorins, most studies so far have found that plexins mediate the signalling outcome. However, it has been proposed that the cytoplasmic domain of membrane-bound semaphorins can also transduce intracellular signals: this is referred to as ‘reverse’ signalling, as opposed to the ‘forward’ signalling activated downstream of the plexin cytoplasmic domain. The paper by Toyofuku et al. published on page 1204 of this issue1 provides the first formal proof that a semaphorin can signal both forward as a lig-and and backwards as a receptor. The authors show that this bidirectional signalling controls guidance of myocardial patterning during car-diac development.

Semaphorins and their receptors — the plex-ins — were originally identified in the nervous system, where they are required to establish the correct neuronal network, steering axon growth cones and dendrites to their final targets. But, consistent with their widespread distribution, their repertoire has since expanded to several non-neural processes, including cardiac and skeletal development, the immune response

and epithelial morphogenesis. More recently, semaphorins have been implicated in tumour growth and metastasis2.

Several functions of semaphorins seem to be accounted for by their ability to either inhibit or induce directional cell motility. Consistent with this, semaphorin and semaphorin receptors are phylogenetically related to scatter factor recep-tors3, a family of transmembrane molecules that control invasive growth. This process is seen during both morphogenesis and cancer progression, when adherent cells scatter and spread to invade the surrounding tissues4.

In a previous study5, the Kikutani group investigated the forward signalling of sema-phorins and showed that in the developing heart the migration of endocardial cells expressing plexin A1 is guided by the adjacent myocardial cells expressing its ligand, Sema6D. They found that the outcome of plexin forward signalling depended on the co-receptor expressed in dif-ferent endocardial cells. Thus, motility of cells expressing the off track kinase (OTK) co-recep-tor is inhibited by Sema6D, whereas motility of cells expressing the vascular endothelial growth factor receptor type 2 (VEGFR2) is stimulated by Sema6D (Fig. 1). OTK and VEGFR2 are not the only possible partners for plexins, as it has been shown that other kinases, such as Met, Her2, Fyn and Fes may also modulate plexin forward signalling6,7.

The Kikutani group now provide strong evidence that semaphorins can also function in ‘reverse gear’, triggering reverse signalling

when bound to plexin A1. In the developing myocardium, plexin A1 is co-expressed with its ligand Sema6D in a subset of proliferating non-motile myocardial cells, forming the ‘compact’ external layer. A sub-population of these cells can switch to a highly motile phenotype and migrate to form the inner ‘trabecular’ layer, in a process that seems to be analogous to the epi-thelial–mesenchymal transition of cells during invasive growth8. This study therefore supports the idea that Sema6D can elicit independent signalling pathways in the two myocardial cell populations: it induces plexin A1 ‘forward’ sig-nalling in cells of the compact layer, whereas in the motile cells that express Sema6D but not plexin A1, it triggers a ‘reverse’ signalling pathway through its own cytoplasmic tail, and generates the trabeculate cell layer.

Thus, Sema6D reverse signalling — activated though an interaction with the extracellular domain of plexin A1 on adjacent cells — seems to provide a ‘go’ signal that kick-starts myo-cardial cell motility. Toyofuku et al. also find that the Abl cytoplasmic tyrosine kinase and cytoskeletal regulators of the Ena/Mena/VASP family are required for this response. They see that activation of Sema6D leads to Abl kinase activation, phosphorylation of Ena and its dis-sociation from the semaphorin tail. One open question, therefore, is whether this mechanism of reverse signalling may be shared among all transmembrane semaphorins. A closer look at the cytoplasmic domain of class 6 semaphorins shows that, although quite long, this region is

Paolo M. Comoglio, Luca Tamagnone and Silvia Giordano are in the Division of Molecular Oncology, IRCC, Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo (Torino), Italy.e-mail: [email protected]




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A second RING to destroy p27Kip1