Uncover A Regulatory Mechanism In Cell Cycle Progression By SCF^βTrCP Induced DYRK1A Degradation And The Roles Of DYRK1A In AML Cell Proliferation

Section IE3 ubiquitin ligase SCFβTrCP mediated the degradation of DYRK1A, a regulatory mechanism in cell cycle progressionBackgroundDown Syndrome (DS) was one of the most common genetic diseases in human. Patients with DS exhibited impaired neural development and most of them would develop early-onset Alzheimer’s Disease (AD). Studies suggested disrupted self-renewal of neural progenitor cells could contribute to the pathology of both DS and AD.DYRK1A, locating in Down Syndrome Critical Region, was considered to participate in the pathology of DS and AD. DYRK1A encoded protein exhibited protein kinase activity, and could phosphorylated several cell cycle regulators, such as CyclinD1, p21 and p27. Studies showed that DYRK1A overexpression could arrest cell cycle and promote differentiation of neural progenitor cells. As DYRK1A exhitited strong gene dosage effect, post-translational modification and regulation was critical to maintain normal level of DYRK1 A. Although DYRK1A played important roles in cell proliferation and differentiation, the expression profile and regulatory mechanism of DYRKlAin cell cycle was still not clarified.Protein complex SCFβTrCP was the unique E3 ligase exhibiting substrate specificity. Subunit βTrCP could specifically bind to D(p)SGXX(p)S sequence of substrates, resulting ubiquitination and degradation through proteasome.SCFβTrCP regulated cell proliferation by controlling degradation of cell cycle regulators, such as CDC25A/B、DEPTOR and Wee.Many studies showed βTrCP expression level was closely correlated with cell cycle stage and exhibited strict regularity, indicating the regulatory importance of SCFβTrCP in cell cycle regulation.ObjectivesThe purpose of this study was to study the expression profile of DYRKIA and βTrCP in cell cycle progression, and the uncover detailed mechanism of SCFβTrCP induced DYRKIA degradation.Methods1. To determine whether ubiquitin-proteasome pathway was involved in DYRK1A degradation. Hek293 cells were transfected with DYRKIA expressing plasmid pDYRK1A-flag-myc, then cells were treated with serial concentrations of lactacystin for 24 hrs or 2.0μM Lac for indicated time. Western blot was used to detect protein level of DYRKIA, and CHX chase assay was applied to determine the DYRKIA degradation rate. Hek293 was co-transfected with pDYRK1A-flag-myc and ubiquitin expressing construct pUbi-his.Ubiquitin or flag antibody were used as pull-down antibody in co-IP assay, and ubiquitinated DYRKIA was detected by western blot.2. To verify whether E3 ligase SCFβTrCP participated in the degradation of DYRK1A. After transfected with pDYRKlA-flag-myc and βTrCP expressing plasmid pβTrCP-flag, Hek293 cells were harvested and co-IP assay was applied to determine the interaction between DYRK1A and βTrCP. BetaTrCP siRNAs were synthesized and co-transfected with pDYRK1A-flag-myc.Forty-eight hours later, DYRKIA protein level was quantified by western blot. Then we constructed the dominant negative inhibitor of Cullinl and Cullin2, which were separatedly co-expressed in Hek293 cells with DYRKIA.Western blot was performed to test the effect of Cullinl or Cullin2 dominant negative inhibitor on DYRKIA protein level.Next we established a PTrCP knocked out Hek293 cell line. Changes of DYRKIA degradation rate was studied using CHX chase assay. After PTrCP was overexpressed or knocked down, ppTrCP-flag and pDYRK1A-flag-myc were co-transfected into Hek293 cells.Ubiquitination of DYRKIA was determined by co-IP assay.3. To confirm which domain controlled DYRK1A degradation. We constructed a series of truncation mutants of DYRK1A. Then CHX chase assay was applied to compare the degradation rate between truncation mutants and wild type DYRK1A.4. To find the βTrCP binding sequence in DYRK1A protein. Wild type DYRK1A or its truncation mutants were co-transfected with pβTrCP-flag into Hek293 cells, and co-IP assay was carried out to detect the interaction with βTrCP. Then we constructed truncation mutants in the N-terminal of DYRK1A. As described above, co-IP assay was used to estimate the ability to interact with βTrCP, and CHX chase assay was performed to determine the degradation rate.5. To identify the degron in DYRK1A protein sequence. We constructed 4 point mutants in which Serine or Threonine was substituted by Alanine. Co-IP assay was used to detect the interaction with PTrCP or ubiquitination ratio, and CHX chase was applied to determine changes in degradation rate. Hek293 cells were co-transfected with pβTrCP-flag and pDYRK1A-flag-myc or DYRK1A N-terminal expressing construct. Cell lysates were treated with λ-PPase and co-IP assay was carried to detect the interaction between (3TrCP and DYRK1A or its N-terminal.6. To explore whether SCFβTrCP could induce DYRK1A protein degradation in cell cycle progression. Hek293 cells were synchronized at G2/M phase by Nocodazole treatment. Cells were then cultured in medium without Nocodazole. In the next 0-24 hrs,cells were collected every 2 hrs. Cell cycle progression was determined by FACS. Western blot was used to detect DYRK1A and βTrCP protein level.Real-time qRT-PCR was performed to estimated DYRK1A mRNA level.BetaTrCP knocked out cells were tansfected with DYRK1A siRNAs expressing vectors, and FACS was performed to detect cell cycle.Results1. DYRK1A was degraded through ubiquitin-proteasome pathway. DYRK1A level was significantly increased in a time and dosage dependent manner after treatment with Lac, and the degradation rate was also markedly decelerated by Lac.In the co-IP assay, ubiquitinated DYRK1A was detected using either ubiquitin antibody or flag antibody as the pull-down antibody.2. E3 ligase SCFβTrCp participated in the degradation of DYRKIA. First we demonstrated that DYRKIA could interact with βTrCP through co-IP assay. When (3TrCP was knocked down in Hek293 cells, the protein level and half-life of DYRKIA were both significantly increased.We also found both dominant negative inhibitors of Cullinl and Cullin2 could remarkably upregulated DYRKIA protein level. Consistent with these results, the ubiquitination ratio of DYRK1A was noticeably reduced or raised when βTrCP was knocked out or overexpressed,respectively.3. The N-terminal of DYRKIA controlled its degradation. We found by CHX chase assay that the degradation rate of DYRKIA C-terminal and mutants with truncated C-terminal or PEST rich region, showed no significance comparing with wild type DYRKIA. However, the N-terminal exhibited extraordinary instability and degraded rapidly in cells.4. The N-terminal 34-71aa of DYRKIA was the binding sequence of βTrCP. We found that PTrCP could bind to the N-terminal of DYRK1A using co-IP assay. Next we constructed several mutants with truncations in the N-terminal. We proved that the N-terminal 34-71aa was the binding sequence of PTrCP. The N-terminal 34-71aa truncation mutant showed prolonged half-life and decreased ubiquitination, providing more evidences to our hypothesis.5. The N-terminal 49SDQQVSALS57 sequence was the degron of DYRKIA. In the CHX chase assay, we found that the degradation of point mutants S49A, S54A and S54A was significantly decelerated compared to wild type DYRKIA. The co-IP assay provided similar results. The interaction between βTrCP and point mutants S49A, S54A or S54A was markedly reduced, and corresponding ubiquitination was also significantly decreased. However,S59A did not show notable changes. Lambda-PPase could remarkably attenuate the binding between PTrCP and DYRK1A or its N-terminal.6. SCFβTrCP regulated DYRK1A protein level in cell cycle progression. Hek293 cells were successfully arrested at G2/M phase after Nocodazole treatment. Cell cycle was recovered upon addition of fresh medium. DYRK1A protein level was significantly decreased at S phase and G2/M phase, and its mRNA level showed little changes during cell cycle progression. BetaTrCP protein exhibited opposite change trend, exhibiting negative correlation with that of DYRK1A. DYRK1A down-regulation could significantly rescued G0/G1 arrest induced by βTrCP knockdown.Conclusions1. E3 ligase SCFβTrCP mediated DYRK1A degradation through ubiquitin-proteasome pathway.2. The DYRK1A N-terminal 49SDQQVSALS57 sequence was the binding sequence of SCFβTrCP.3. Regulation of DYRK1A degradation by SCFβTrCP was essential for cell cycle progression.Section ⅡDYRK1A effected on proliferation and chemoresistance of AML cells by down-regulating c-MycBackgroundAcute myeloid leukemia (AML), a malignancy caused by abnormal proliferation and accumulation of hematopoietic progenitor cells, was the most common leukemia type in human adults. Although the treatment of AML had been rapidly improved, little AML patients were cured and five-year overall survival was still poor. The biggest obstacles for AML therapy was lack of targeting drugs and drug resistance.It was great significance to explore underlying pathological mechanism of AML, providing more evidences for developing new therapy strategies. However, the molecular mechanisms for AML were still not clear. Besides genetic mutations, aberrant expression of tumor suppressor genes and oncogenes was also thought to play important roles in AML genesis.DYRK1A, locating on the Down Syndrome Critical Region of chromosome 21, was involved in the pathology of Down Syndrome. The risk of solid tumors and several types of leukemia was significantly reduced, implying the anti-tumor effects of DYRK1 A. DYRK1A, a protein kinase, could phosphorylate serine or threonine residues of its substrates, several of which were closely related to tumor genesis, metastasis and drug resistance, cell proliferation and differentiation. However, the effect and mechanism of DYRK1A in AML was still needed to be explored.c-Myc was a well known oncogene. Its encoded protein mainly involved in cell proliferative and differentiational regulation. C-Myc was abnormally overexpressed in pathological conditions, resulting in shortened cell cycle, differentiational inhibition and malignant proliferation. Studies showed that c-Myc protein was overexpressed in AML patients, indicating its close relationship with c-Myc.C-Myc protein level decreased after treatment and raised again upon relapse,suggesting that it was critical to reduce c-Myc protein in AML treatment. Besides transcription and translation, c-Myc protein level was regulated through degradation. Degradation of c-Myc was mainly induced by ubiquitin-proteasome pathway, in which phosphorylation played important roles, suggesting the necessity to discover new c-Myc kinases for fully understanding the pathological mechanism of AML.ObjectivesThe aim of this study was to explore the expression profile of DYRK1 A in AML patients and the effect of DYRK1 A in AML cell proliferation and drug resistance. We would further explore whether DYRK1 A could regulate c-Myc protein and related molecular mechanisms. Our study would provide more evidences to develop new targeting drugs for AML therapy.Methods1. To study the expression profile of DYRK1A in AML patients. Clinical samples were collected from 55 newly Newly-diagnosed and relapsed/refractory AML patients. Mononuclear cells were prepared using Ficoll-Hypaque. RNA was isolated utilizing Trizol reagent and reverse transcription were performed in vitro. DYRK1 A mRNA level was determined by SYBR Green real-time qRT-PCR.2. To identify the effect of DYRK1A on AML cell proliferation. The expression level of DYRK1 A mRNA was evaluated by SYBR Green real-time qRT-PCR after cells were infected with DYRK1 A lentiviral particles. Then we performed MTT assay to determine cell proliferation rate and flow cytometry to test cell cycle changes. Besides that, we also detected the protein level of cell cycle regulators p21, cyclin D1 and CDK2. To eliminate the possibility that cell apoptosis might also impact cell viability, cells were stained by annexin V-PE/7-AAD and subjected for FACS analysis.3. To determine whether DYRK1A could regulate c-Myc and corresponding molecular pathways. Overexpression of DYRK1 A in AML cells was verified by SYBR Green real-time qRT-PCR and western blot. To examine whether DYRK1A could promote the degradation of c-Myc, the degradation rate of c-Myc was determined by CHX chase assay. The phosphorylation status of Ser62 and Thr58 residues, which regulated the degradation of c-Myc, was also detected.To provide more evidences that DYRK1 A could phosphorylate c-Myc, co-IP was utilized to study the interaction between DYRK1A and c-Myc.4. To verify whether DYRK1A induced AML cell proliferative inhibition functioned through mediating c-Myc degradation. We down-regulated c-Myc expression using RNAi construct. The mRNA level of cell cycle regulators cyclin D1,CDK2 and p21 was evaluated. Then we explored that whether the decrease of cell cyclin D1 or the increase of cell proliferative rate and p21 induced by DYRKIA overexpression could be rescued by c-Myc overexpression.5. To test the role of DYRKIA in AML drug resistance. DYRKIA was overexpressed in HL-60/ADM cells. After treated with gradient dilutions of doxorubicin for 72 hrs, cell viability was measured by MTT assay, and cell apoptosis was determined by Annexin V-PE/7-AAD staining.Results1. DYRKIA mRNA level was abnormally lower in AML patients. In real-time qRT-PCR assay, we found DYRKIA mRNA level in AML patients was significantly lower than that of normal control. The reduced DYRKIA mRNA level was also found in newly-diagnosed AML patients compared with newly-diagnosed AML patients.2. DYRK1A significantly inhibited AML cell proliferation. The proliferation of HEL, HL-60 and NB4 was markedly suppressed by overexpression of DYRKIA, which was further supported by the observation that the proportion of cells in G0/G1 phase was significantly increased, while simultaneously decreased the ratio of cells undergoing S phase. In accordance with this, p21 protein level was markedly upregulated, while cyclin D1 and CDK2 protein level was significantly down-regulated. The FACS results showed that DYRK1A had little effect on the apoptosis of AML cells, making our results more reliable.3. DYRK1A promoted c-Myc degradation through phosphorylation. We observed that DYRK1A overexpression could noticeably upregulated the protein level other than mRNA level of c-Myc. We further found that DYRKIA accelerated the degradation of c-Myc by promoting the phosphorylation of Ser62 and Thr58 residues, which had been reported to be responsible for c-Myc proteolysis. The co-IP result that DYRK1A could interact with c-Myc provided further evidences to support our study.4. DYRK1A functioned through c-Myc decrement to inhibit AML cell proliferation. In c-Myc knocked down HEL cells, p21 mRNA level was significantly increased by 105.36± 42.94%, while that of cyclin D1 was markedly decreased by 49.53±2.43%. On the other hand, c-Myc overexpression could effectively rescued the proliferative inhibition, p21 up-regulation and cyclin D1 attenuation induced by DYRK1 A overexpression.5. DYRK1 A enhanced drug sensitivity of AML cells. The doxorubicin IC50 of HL-60/ADM-DYRK1Acell line, which overexpressed DYRK1 A, was 0.9389±0.063 mg/L, significantly lower than that of control cells (2.3144±0.58299 mg/L). After treatment of doxorubicin, HL-60/ADM-DYRK1A cells exhibited higher ratio of apoptosis, which was consistent with the results of IC50 measurement.Conclusions1. DYRK1 A antagonized the proliferation of AML cells.2. DYRK1 A promoted c-Myc degradation by phosphorylating its Ser62 residue.3. The anti-tumor effects of DYRK1 A in AML functioned partially through mediating the degradation of c-Myc.