Philippe Coulombe

p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis

The activity of the cyclin‐dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild‐type p27 and a p27(S10D)‐phospho‐mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin‐dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B‐mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half‐life than wild‐type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin–Cdk2 complexes.

Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors

ABSTRACT As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67phox. We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

ABSTRACT Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

Rapid Turnover of Extracellular Signal-Regulated Kinase 3 by the Ubiquitin-Proteasome Pathway Defines a Novel Paradigm of Mitogen-Activated Protein Kinase Regulation during Cellular Differentiation

ABSTRACT Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G1 arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.

Involvement of the IκB Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1−/− mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IκB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.

Phosphorylation of Skp2 regulated by CDK2 and Cdc14B protects it from degradation by APCCdh1 in G1 phase

The p27Kip1 ubiquitin ligase receptor Skp2 is often overexpressed in human tumours and displays oncogenic properties. The activity of SCFSkp2 is regulated by the APCCdh1, which targets Skp2 for degradation. Here we show that Skp2 phosphorylation on Ser64/Ser72 positively regulates its function in vivo. Phosphorylation of Ser64, and to a lesser extent Ser72, stabilizes Skp2 by interfering with its association with Cdh1, without affecting intrinsic ligase activity. Cyclin‐dependent kinase (CDK)2‐mediated phosphorylation of Skp2 on Ser64 allows its expression in mid‐G1 phase, even in the presence of active APCCdh1. Reciprocally, dephosphorylation of Skp2 by the mitotic phosphatase Cdc14B at the M → G1 transition promotes its degradation by APCCdh1. Importantly, lowering the levels of Cdc14B accelerates cell cycle progression from mitosis to S phase in an Skp2‐dependent manner, demonstrating epistatic relationship of Cdc14B and Skp2 in the regulation of G1 length. Thus, our results reveal that reversible phosphorylation plays a key role in the timing of Skp2 expression in the cell cycle.

Activation loop phosphorylation of the atypical MAP kinases ERK3 and ERK4 is required for binding, activation and cytoplasmic relocalization of MK5

Mitogen‐activated protein (MAP) kinases are typical examples of protein kinases whose enzymatic activity is mainly controlled by activation loop phosphorylation. The classical MAP kinases ERK1/ERK2, JNK, p38 and ERK5 all contain the conserved Thr‐Xxx‐Tyr motif in their activation loop that is dually phosphorylated by members of the MAP kinase kinases family. Much less is known about the regulation of the atypical MAP kinases ERK3 and ERK4. These kinases display structural features that distinguish them from other MAP kinases, notably the presence of a single phospho‐acceptor site (Ser‐Glu‐Gly) in the activation loop. Here, we show that ERK3 and ERK4 are phosphorylated in their activation loop in vivo. This phosphorylation is exerted, at least in part, in trans by an upstream cellular kinase. Contrary to classical MAP kinases, activation loop phosphorylation of ERK3 and ERK4 is detected in resting cells and is not further stimulated by strong mitogenic or stress stimuli. However, phosphorylation can be modulated indirectly by interaction with the substrate MAP kinase‐activated protein kinase 5 (MK5). Importantly, we found that activation loop phosphorylation of ERK3 and ERK4 stimulates their intrinsic catalytic activity and is required for the formation of stable active complexes with MK5 and, consequently, for efficient cytoplasmic redistribution of ERK3/ERK4‐MK5 complexes. Our results demonstrate the importance of activation loop phosphorylation in the regulation of ERK3/ERK4 function and highlight differences in the regulation of atypical MAP kinases as compared to classical family members. J. Cell. Physiol. 217: 778–788, 2008.

p107 inhibits G1 to S phase progression by down-regulating expression of the F-box protein Skp2

Cell cycle progression is negatively regulated by the pocket proteins pRb, p107, and p130. However, the mechanisms responsible for this inhibition are not fully understood. Here, we show that overexpression of p107 in fibroblasts inhibits Cdk2 activation and delays S phase entry. The inhibition of Cdk2 activity is correlated with the accumulation of p27, consequent to a decreased degradation of the protein, with no change of Thr187 phosphorylation. Instead, we observed a marked decrease in the abundance of the F-box receptor Skp2 in p107-overexpressing cells. Reciprocally, Skp2 accumulates to higher levels in p107 −/− embryonic fibroblasts. Ectopic expression of Skp2 restores p27 down-regulation and DNA synthesis to the levels observed in parental cells, whereas inactivation of Skp2 abrogates the inhibitory effect of p107 on S phase entry. We further show that the serum-dependent increase in Skp2 half-life observed during G1 progression is impaired in cells overexpressing p107. We propose that p107, in addition to its interaction with E2F, inhibits cell proliferation through the control of Skp2 expression and the resulting stabilization of p27.

Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2

Skp2 is the substrate binding subunit of the SCFSkp2 ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APCCdh1, which targets Skp2 for degradation in early G1 and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCFSkp2 ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.

Dual-tag prokaryotic vectors for enhanced expression of full-length recombinant proteins.

The ability to express and purify proteins in large amounts through recombinant DNA technology has enabled significant advances in the biomedical sciences. Several commercially available or home-made vectors allow expression and easy purification of recombinant proteins in Escherichia coli. Generally, such vectors are designed to produce fusion proteins containing a molecular tag at the N-terminal end. Calmodulin, streptavidin, maltose binding protein, thioredoxin, hexahistidine ðHis6Þ, and glutathione S-transferase (GST) are examples of tags chosen for their high binding affinity toward specific ligands [7]. However, this procedure also has inherent limitations. In some cases, proteins overexpressed in E. coli form insoluble aggregates that are difficult to solubilize or are found mainly as truncated forms [1]. Purification of recombinant fusion proteins sometimes requires several chromatography steps and renaturation protocols. These problems are particularly evident for large proteins, for which yields of purification are frequently very low and preclude further analysis or use of the recombinant protein. Although several procedures have been developed to improve protein solubility in E. coli [1,2,6,8], less progress has been made to overcome the problem of truncated protein expression due to proteolysis, intrinsic instability, or premature translation termination caused by codon usage bias [4]. Here we describe the construction of novel dual-tag prokaryotic expression vectors that allow for enrichment in full-length recombinant proteins and facilitate their subsequent purification by sequential chromatography on metal and glutathione affinity columns. Extracellular signal-regulated kinase 3 (ERK3) is a member of the MAP kinase family of serine/threonine kinases [5]. In the course of our studies on this enzyme, we experienced problems in producing good yields of full-length GST fusion constructs of ERK3 using conventional prokaryotic expression systems. To circumvent these problems, we created a novel set of dual-tag prokaryotic expression vectors, pHGST.1 and pHGST. 2T, that were engineered to produce N-terminal His6and C-terminal GST-tagged fusion proteins (Fig. 1). In theory, the sequential purification of recombinant proteins on glutathione and metal affinity resins should: (1) allow for enrichment in full-length protein and (2) facilitate the purification process and increase the purity of the final product. pHGST vectors were constructed by first subcloning the two annealed oligonucleotides 50 CTA GCA TGA ATT CGG GAT CCA TGG GTC GAC TCG AGC TCG GAA 30 and 50 AGC TTT CCG AGC TCG AGT CGA CCC ATG GAT CCC GAA TTC ATG 30 into the NheI/HindIII sites of pRSET-A (Invitrogen Life Technologies, Burlington, Canada) to generate pRSET-MCS. The GST coding sequence was amplified by PCR using pGEX-KG [3] as template with the following oligonucleotides: 50 GCC GAG CTC TCC CCT ATA CTA GGT TAT TGG 30 and 50 CCC AAG CTT TTA ATC CGA TTT TGG AGG ATG GTC 30. The amplicon was then digested with SstI/ HindIII and subcloned into the SstI/HindIII sites of pRSET-MCS to yield pHGST.1. The pHGST.2T vector was obtained by ligation of the following annealed oligonucleotides into NcoI/XhoI-digested pHGST.1: 50 CAT GGC GGC CGC CTC GAG TCT GGT TCC Analytical Biochemistry 310 (2002) 219–222