Disruption of Myc-tubulin interaction by hyperphosphorylation of c-Myc during mitosis or by constitutive hyperphosphorylation of mutant c-Myc in Burkitt's lymphoma

Somatic mutations at Thr-58 of c-Myc have been detected in Burkitt's lymphoma (BL) tumors and have been shown to affect the transforming potential of the Myc oncoprotein. In addition, the N-terminal domain of c-Myc has been shown to interact with microtubules in vivo, and the binding of c-Myc to alpha-tubulin was localized to amino acids 48 to 135 within the c-Myc protein. We demonstrate that c-Myc proteins harboring a naturally occurring mutation at Thr-58 from BL cell lines have increased stability and are constitutively hyperphosphorylated, which disrupts the in vivo interaction of c-Myc with alpha-tubulin. In addition, we show that wild-type c-Myc-alpha-tubulin interactions are also disrupted during a transient mitosis-specific hyperphosphorylation of c-Myc, which resembles the constitutive hyperphosphorylation pattern of Thr-58 in BL cells.     

c-Jun-NH2 kinase (JNK) contributes to the regulation of c-Myc protein stability

In accord with the central role c-Myc plays in control of cell growth and death, the stability of this protein is tightly regulated. Although the NH2-terminal domain of c-Myc has been implicated in the regulation of its stability, c-Myc-S, which lacks this domain, is equally unstable, pointing to the role of additional domains in the regulation of c-Myc stability. Our former studies revealed that amino acids (aa) 127-189 of c-Myc are responsible for stress-induced stability of the c-Myc protein. This region of c-Myc shares homology with the delta domain of c-Jun, which is required for JNK association and subsequent targeting of c-Jun for ubiquitination under non-stressed growth conditions. Here we demonstrate that JNK associates with, and mediates, c-Myc ubiquitination and degradation. Addition of JNK increased the degree of c-Myc ubiquitination in in vitro ubiquitination reactions. Increased c-Myc stability following MEKK1/JNK stimuli is abolished upon mutation within the delta-like domain of c-Myc (aa 166-181), as well as deletion of aa 127-189. Significantly, inhibition of JNK expression via small interfering RNA increased c-Myc protein expression. Similarly, squelching JNK association with c-Myc by overexpression of a peptide corresponding to aa 127-189 of c-Myc increased endogenous c-Myc stability and elevated the fraction of cells within the G2/M phase of the cell cycle. In all, these findings point to the contribution of JNK to the regulation of c-Myc protein stability under normal growth conditions.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.     

ASK1-signaling promotes c-Myc protein stability during apoptosis

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.     

FBXO32 Targets c-Myc for Proteasomal Degradation and Inhibits c-Myc Activity

FBXO32 (MAFbx/Atrogin-1) is an E3 ubiquitin ligase that is markedly up-regulated in muscle atrophy. Although some data indicate that FBXO32 may play an important role in tumorigenesis, the molecular mechanism of FBXO32 in tumorigenesis has been poorly understood. Here, we present evidence that FBXO32 targets the oncogenic protein c-Myc for ubiquitination and degradation through the proteasome pathway. Phosphorylation of c-Myc at Thr-58 and Ser-62 is dispensable for FBXO32 to induce c-Myc degradation. Mutation of the lysine 326 in c-Myc reduces c-Myc ubiquitination and prevents the c-Myc degradation induced by FBXO32. Furthermore, overexpression of FBXO32 suppresses c-Myc activity and inhibits cell growth, but knockdown of FBXO32 enhances c-Myc activity and promotes cell growth. Finally, we show that FBXO32 is a direct downstream target of c-Myc, highlighting a negative feedback regulation loop between c-Myc and FBXO32. Thus, FBXO32 may function by targeting c-Myc. This work explains the function of FBXO32 and highlights its mechanisms in tumorigenesis.     

Role of Pirh2 in mediating the regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2(-/-) mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.     

The c-Myc transactivation domain is a direct modulator of apoptotic versus proliferative signals

We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals.     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.     

Deubiquitinating c-Myc: USP36 steps up in the nucleolus

Ubiquitination plays a key and complex role in the regulation of c-Myc stability, transactivation, and oncogenic activity. c-Myc is ubiquitinated by a number of ubiquitin ligases (E3s), such as SCF^Fbw7 and SCF^Skp2. Depending on the E3s, ubiquitination can either positively or negatively regulate c-Myc levels and activity. Meanwhile, c-Myc ubiquitination can be reversed by deubiquitination. An early study showed that USP28 deubiquitinates c-Myc via interacting with Fbw7α whereas a recent study reveals that USP37 deubiquitinates c-Myc independently of Fbw7 and c-Myc phosphorylation. Consequently, both USP28 and USP37 stabilize c-Myc and enhance its activity. We recently found the nucleolar USP36 as a novel c-Myc deubiquitinase that controls the end-point of c-Myc degradation pathway in the nucleolus. Here we briefly review the current understanding of ubiquitination and deubiquitination regulation of c-Myc and further discuss the USP36-c-Myc regulatory pathway.     

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.     

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.     

c-Myc蛋白与DNA-PKcs作用位点的鉴定

DNA-PK复合物由Ku蛋白和DNA依赖蛋白激酶催化亚基(DNA-PKcs)组成,DNA-PKcs属于PI3K相关激酶家族成员.我们前期工作发现,DNA-Kcs沉默后,c-Myc的稳定性下降,且二者存在相互作用.为进一步确定c-Myc蛋白与DNA-PKcs相互作用位点,本研究利用原核表达系统活动了c-Myc及其截短体蛋白,利用GST pull-down技术结合Western印迹法,发现c-Myc蛋白294～370位氨基酸与DNA-PKcs存在相互作用.在细胞内表达GFP-c-Myc各截短体蛋白,发现294～370位氨基酸是c-Myc蛋白降解必需的.利用免疫荧光技术,发现DNA-PKcs与c-Myc蛋白有相同的细胞亚定位,进一步表明两者在生物学功能上具有相关性.有文献报道294～370位氨基酸是乙酰转移酶p300的底物,此位点的乙酰化导致c-Myc的降解.本实验结果提示,c-Myc蛋白的294～370位氨基酸与DNA-PKcs结合,可能阻止了乙酰转移酶p300的结合,从而达到提高c-Myc蛋白稳定性的作用.