Siah1/SIP regulates p27kip1 stability and cell migration under metabolic stress

p27^kip1 has been implicated in cell cycle regulation, functioning as an inhibitor of cyclin-dependent kinase activity. In addition, p27 was also shown to affect cell migration, with accumulation of cytoplasmic p27 associated with tumor invasiveness. However, the mechanism underlying p27 regulation as a cytoplasmic protein is poorly understood. Here we show that glucose starvation induces proteasome-dependent degradation of cytoplasmic p27, accompanied by a decrease in cell motility. We also show that the glucose limitation-induced p27 degradation is regulated through an ubiquitin E3 ligase complex involving Siah1 and SIP/CacyBP. SIP^-/- embryonic fibroblasts have increased levels of cytosolic p27 and exhibit increased cell motility compared to wild-type cells. These observations suggest that the Siah1/SIP E3 ligase complex regulates cell motility through degradation of p27.     

Structural analysis of Siah1-Siah-interacting protein interactions and insights into the assembly of an E3 ligase multiprotein complex

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.     

p27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation

The cyclin-dependent kinase inhibitor p27^Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome–lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27^−/− MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27^+/+ MEFs and impaired autophagy flux, similar to that observed in p27^−/− cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism.Subject terms: Tumour-suppressor proteins, Macroautophagy     

KPC1 expression and essential role after acute spinal cord injury in adult rat

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27^kip1 at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27^kip1 following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27^kip1 and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27^kip1 expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27^kip1 and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27^kip1; this event may be involved in the proliferation of astrocytes after SCI.     

Substituting Threonine 187 with Alanine in p27Kip1 Prevents Pituitary Tumorigenesis by Two-Hit Loss of Rb1 and Enhances Humoral Immunity in Old Age

p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases. Inhibiting p27 protein degradation is an actively developing cancer therapy strategy. One focus has been to identify small molecule inhibitors to block recruitment of Thr-187-phosphorylated p27 (p27T187p) to SCF^Skp2/Cks1 ubiquitin ligase. Since phosphorylation of Thr-187 is required for this recruitment, p27T187A knockin (KI) mice were generated to determine the effects of systemically blocking interaction between p27 and Skp2/Cks1 on tumor susceptibility and other proliferation related mouse physiology. Rb1^+/− mice develop pituitary tumors with full penetrance and the tumors are invariably Rb1^−/−, modeling tumorigenesis by two-hit loss of RB1 in humans. Immunization induced humoral immunity depends on rapid B cell proliferation and clonal selection in germinal centers (GCs) and declines with age in mice and humans. Here, we show that p27T187A KI prevented pituitary tumorigenesis in Rb1^+/− mice and corrected decline in humoral immunity in older mice following immunization with sheep red blood cells (SRBC). These findings reveal physiological contexts that depend on p27 ubiquitination by SCF^Skp2-Cks1 ubiquitin ligase and therefore help forecast clinical potentials of Skp2/Cks1-p27T187p interaction inhibitors. We further show that GC B cells and T cells use different mechanisms to regulate their p27 protein levels, and propose a T helper cell exhaustion model resembling that of stem cell exhaustion to understand decline in T cell-dependent humoral immunity in older age.     

TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth

We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27^kip1(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.     

Sec6 regulated cytoplasmic translocation and degradation of p27 via interactions with Jab1 and Siah1

p27 has essential roles in cellular proliferation and migration, and reduced or cytoplasmic p27 is associated with poor clinical outcomes in a variety of human tumours. Jun activation domain-binding protein (Jab1)/constitutive photomorphogenic-9 signalosome 5 (CSN5) directly interacts with p27 promoting its translocation and cytoplasmic degradation. Sec6 is a component of the exocyst complex. Recently, several studies revealed that Sec6 has specific functions in migration, adhesion, and cell differentiation. However, how Sec6 is involved in the regulation of cell cycle progression is unknown. The present study shows that Sec6 regulates cytoplasmic translocation of p27 through p27 phosphorylation at Thr157, thereby promoting p27 degradation in the cytoplasm via interaction with Jab1 and Siah1 and suppressing cell cycle progression.     

Regulating the stability and localization of CDK inhibitor p27Kip1 via CSN6-COP1 axis

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27^Kip1 stability, and that COP1 is a negative regulator of p27^Kip1. Ectopic expression of CSN6 can decrease the expression of p27^Kip1, while CSN6 knockdown leads to p27^Kip1 stabilization. Mechanistic studies show that CSN6 interacts with p27^Kip1 and facilitates ubiquitin-mediated degradation of p27^Kip1. CSN6-mediated p27 degradation depends on the nuclear export of p27^Kip1, which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double strand break repair, and stress-induced cell-cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27, and show that Cul4A-DDB1^DDB2-Artemis regulates G₁ phase cell cycle progression in mammalian cells.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double-strand break repair and stress-induced cell cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27 and show that Cul4A-DDB1^DDB2-Artemis regulates G₁-phase cell cycle progression in mammalian cells.Key words: artemis, DDB2, p27, Cul4A-DDB1, ubiquitylation     

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21^Waf1/Cip1. We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53^S18P) and targets it for degradation in low-dose-UV-irradiated cells. DDB2^−/− mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53^S18P. Accumulation of p53^S18P in DDB2^−/− MEFs causes higher expression p21^Waf1/Cip1. We show that the increased expression of p21^Waf1/Cip1 is the cause NER deficiency in DDB2^−/− cells because deletion or knockdown of p21^Waf1/Cip1 reverses their NER-deficient phenotype. p21^Waf1/Cip1 was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21^Waf1/Cip1 was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2.     

Increased Cytoplasmic Localization of p27kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

The role of p27^kip1 in Chronic Myeloid Leukemia (CML) has been well studied in relation to its function as a cell cycle inhibitor. However, its cytoplasmic function especially in CML remains to be seen. We studied the localization of p27^kip1 and its function during the progression of CML from chronic to blast phase. Our investigations revealed an increased localization of p27^kip1 in the cytoplasm of CD34⁺ cells in the blast phase compared to chronic phase. Cytoplasmic p27^kip1 was found to modulate RhoA activity in CD34⁺ stem and progenitor cells. Further, RhoA activity was shown to be dependent on cytoplasmic p27^kip1 which in turn was dependent on p210^Bcr-Abl kinase activity. Interestingly, RhoA activity was observed to affect cell survival in the presence of imatinib through the SAPK/JNK pathway. Accordingly, inhibition of SAPK/JNK pathway using SP600125 increased apoptosis of K562 cells in presence of imatinib. Our results, for the first time, thus reveal a crucial link between cytoplasmic p27^kip1, RhoA activity and SAPK/JNK signalling. To this effect we observed a correlation between increased cytoplasmic p27^kip1, increased RhoA protein levels, decreased RhoA-GTP levels and increased SAPK/JNK phosphorylation in blast phase CD34⁺ cells compared to chronic phase CD34⁺ cells.     

Loss of p27kip1 suppresses the myocardial senescence caused by estrogen deficiency

Estrogen deficiency accelerates the aging process and increases the risk of developing cardiovascular disease (CVD). Apoptosis is one of the important mechanisms of aging. p27^kip1 is a cyclin-dependent kinase inhibitor that can regulate cell cycle, apoptosis, and cell motility. p27^kip1 overexpression can inhibit cell cycle and increase apoptosis so it has been considered as a marker of aging. In the present study, bilateral ovariectomy (OVX) was performed as a model for menopause in wild-type (WT) and p27^kip1 knockout (KO) mice to assess the effects of p27^kip1 loss in myocardial aging caused by estrogen deficiency. We found that myocardial fibrosis and heart weight/body weight ratio of mice in the OVX group and p27^kip1 KO group were significantly increased. Echocardiography showed that the left ventricular diameter and volume of the WT OVX group increased significantly and the cardiac function decreased. However, there was no significant difference in the results of echocardiography between the two p27^kip1 KO groups. The aging and apoptosis indexes in OVX group were increased significantly, However, the indexes in p27^kip1 KO mice were decreased. The expression of antioxidant indexes in OVX group was decreased significantly and p27^kip1 KO can improve the antioxidant ability. These results provided that estrogen deficiency increased oxidative stress and apoptosis, accelerated aging of heart. p27^kip1 KO can partly delay the aging and apoptosis of heart through upregulated antioxidant enzymes.     

Dynamic Changes of p27<Superscript>kip1</Superscript> and Skp2 Expression in Injured Rat Sciatic Nerve

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27^kip1. Previous reports have showed that p27^kip1 played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27^kip1 and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27^kip1. By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27^kip1 levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27^kip1 and Skp2 likely play an important role in peripheral nerve injury and regeneration. Ai-Guo Shen and Shu-Xian Shi contributed equally to this work.     

USP19 Deubiquitinating Enzyme Supports Cell Proliferation by Stabilizing KPC1, a Ubiquitin Ligase for p27Kip1

p27^Kip1 is a cyclin-dependent kinase inhibitor that regulates the G₁/S transition. Increased degradation of p27^Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCF^Skp2 ubiquitinate p27^Kip1 in G₁ and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27^Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G₀/G1 to S phase, and accumulated p27^Kip1. This increase in p27^Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27^−/− cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G₁ to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.The ubiquitin proteasome pathway plays essential roles in regulating the cell cycle. The best-defined functions of this pathway in cell cycle regulation are those mediated by the multisubunit ubiquitin protein ligases SKP1-CUL1-F-box (SCF) and the anaphase-promoting complex/cyclosome (APC) (reviewed in reference 25). The functions of the APC in the cell cycle are predominant at the mitosis-anaphase transition, while the activities of SCF-type ubiquitin protein ligase complexes are involved at various steps of the cycle (reviewed in references 21 and 25). One of the best-defined functions of the SCF is mediated by SCF^skp2, which plays a vital role in regulating the G₁-S transition by ubiquitinating the cyclin-dependent kinase inhibitor p27^Kip1, thereby targeting it for degradation by the proteasome (3, 31, 32).The central role of p27^Kip1 in restricting cell proliferation is demonstrated by the fact that mice lacking the p27^Kip1 gene manifest increased body and organ weights and develop pituitary adenomas (6, 13, 18). In addition, the results of clinical studies suggest that low p27^Kip1 levels are associated with increased aggressivity of tumors (1, 28). Unlike the case with the p53 or Rb tumor suppressors, mutation or deletion of p27^Kip1 in tumors is rare. Rather, its deregulation in human tumors is due mainly to reduced protein levels, mediated in large part by increased proteolysis (reviewed in reference 21). In support of this, the low p27^Kip1 levels seen in tumors are associated with increased levels of Skp2, the substrate recognition subunit of the SCF^skp2 ligase. The loss of Skp2 in mice results in p27^Kip1 accumulation, and cells from Skp2^−/− animals contain enlarged nuclei with polyploidy and multiple centrosomes. They also show a reduced growth rate and increased apoptosis (19). Many of the cellular phenotypes observed in Skp2^−/− mice disappear in Skp2^−/− p27^−/− double-mutant mice (14, 20). Thus, the oncogenic nature of Skp2 is largely due to its ability to mediate p27^Kip1 degradation.In spite of the clear role of SCF^skp2 in mediating the ubiquitination and degradation of p27^Kip1, the downregulation of this cyclin-dependent kinase inhibitor proceeds normally in lymphocytes isolated from Skp2^−/− mice (8, 21). In addition, in the normal cell cycle, p27^Kip1 is also degraded in G₁, before the expression of Skp2, which occurs in early S phase (8, 9, 36). Also, p27^Kip1 is exported from the nucleus to the cytoplasm in G₁, whereas Skp2 is localized in the nucleus (9, 26). These observations suggest the existence of another pathway for the degradation of p27^Kip1. Indeed, KPC (Kip1 ubiquitination-promoting complex) was subsequently identified as a novel cytoplasmic ligase complex that interacts with and ubiquitinates p27^Kip1 (10). KPC consists of two subunits: KPC1, a 140-kDa RING-finger domain-containing protein, and KPC2, a 50-kDa protein containing a ubiquitin-like domain and two ubiquitin-associated domains.Although the role of ubiquitin protein ligases in the cell cycle has received considerable attention, fewer data are available regarding the roles of deubiquitinating enzymes in the cell cycle (22). We recently described USP19 as a deubiquitinating enzyme that is induced in skeletal muscle atrophying in response to numerous catabolic stimuli. To study its function, we used RNA interference to explore the consequences of depletion of this enzyme in cultured muscle cells. Our early studies indicated that the loss of USP19 interfered with the growth of L6 myoblasts. We have observed similar effects in FR3T3 fibroblasts and have explored the underlying mechanisms of this growth defect.