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And C-Terminal Domains of Src Regulate Distinct Roles Of Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Acetylation within the N- and C-terminal domains of Src regulate distinct roles of STAT3-mediated tumorigenesis Chao Huang1, 3, 6 *, Zhe Zhang2, 6, Lihan Chen3, 6, Hank W. Lee3, Marina K. Ayrapetov4, Ting C. Zhao4, Yimei Hao3, Jinsong Gao4, Chunzhang Yang5, Gautam U. Mehta5, Zhengping Zhuang5, Xiaoren Zhang3, Guohong Hu3, and Y. Eugene Chin1, 3 * 1 Translation Medicine Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200025, China 2 Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China 3 Institute of Health Sciences, Chinese Academy of Sciences and Shanghai Jiaotong University School of Medicine, 320 Yueyang Road, Shanghai 200031, China 4 Departments of Surgery and Medicine, Brown University School of Medicine-Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903 USA 5 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland 20892, USA 6 Co-first authors * Correspondence: [email protected] and [email protected] Running tittle: Regulation of Src-STAT3 signaling pathway by acetylation Keywords: c-Src; STAT3; acetylation; EGF; tumorigenesis Grant Support: This work was supported by grants from the National Natural Science Foundation of China (81230059 to Y.E.Chin, 81672804 to C.Huang, 81402097 to Z.Zhang), Ministry of Science and Technology of China (2015CB910402 to C.Huang). Disclosure of Potential Conflicts of Interest Downloaded from cancerres.aacrjournals.org on September1 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. No potential conflicts of interest were disclosed. Significance CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity Downloaded from cancerres.aacrjournals.org on September2 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Post-translational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions: myristoylation of G2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylated an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylated the C- terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activated transcriptionally active dimers of STAT3. Moreover, acetyl- Src translocated into nuclei where it formed the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Introduction Src family kinases are cell membrane-associated non-receptor protein tyrosine kinases that coordinate various cellular events including differentiation, adhesion, and migration. Aberrant Src kinase activity has been widely implicated in cancer development, progression and metastasis (1, 2). Functionally, Src has N-terminal and C-terminal regions of similar size. The human c-Src N-terminal region is comprised of a unique domain (UD), an SH3 domain, and an SH2 domain while the C-terminal region contains a catalytic domain (CD) followed by a regulatory tail (2). Src Downloaded from cancerres.aacrjournals.org on September3 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. associates with the cytoplasmic membrane, which depends on myristoylation of the N- terminal glycine residue (G2) mediated by N-myristoyltransferase (Nmt) and positively charged residues within UD that interact with the overall negatively charged cell membrane (3). Cell membrane association is required for cell transformation, whereas dissociation permits Src to be targeted to other cellular compartments (4, 5). c-Src catalytic activity has been detected in the cytoplasm but also in the endoplasmic reticulum (ER), mitochondria and nucleus (6-8). c-Src is inhibited by the C-terminal Src kinase (Csk) that phosphorylates Y527 (in chicken) or Y530 (in human) and renders Src catalytically inactive as a result of intramolecular interactions between the C- terminal tail and the SH2 domain (9). c-Src catalytic activation needs conformational changes within the catalytic domain: the orientation of two lobes of catalytic domain; rearrangement of the activation loop; and movement of the helical interface between the two lobes into the catalytic cleft. Global phosphorylation of c-Src may cause this conformational change and augment its kinase activity (10). In cancer cells, c-Src is activated by growth factors, and leads to phosphorylation and activation of oncogenic substrates including STAT3 (11). However, it remains unclear how c-Src regulates down-stream targets in response to growth factor treatment. In this study, we report that c-Src global phosphorylation is induced by CBP, an acetyltransferase in the cells. We observe that after EGF stimulation, unique domain and catalytic domain of c-Src are acetylated by CBP, which results in c-Src dissociation from plasma membrane and recruitment and activation of STAT3. Importantly, c-Src forms a protein kinase-transcription factor enhanceosome with STAT3 in the nuclei where c-Src promotes STAT3 activity to up-regulate oncogene expression in cancer cells. Downloaded from cancerres.aacrjournals.org on September4 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Cell Culture and Reagents NIH3T3, HEK293T and MCF-7 cells were purchased from ATCC (Oct, 2010), Src-/- SYF and control (Src introduced) SYF cells from DC Flynn (Apr, 2002), U3A cells from and GR Stark (Feb, 1996), and cbp-/- MEF were cultured in DMEM medium containing 10% fetal bovine serum, 2 μM glutamine, and 100 U/ml each of penicillin and streptomycin by Invitrogen (Carlsbad, CA). MCF-7 and U3A cells were authenticated using short tandem repeat (STR) profiling analysis by Suzhou Genetic Testing Biotechnology Co. Ltd. HEK293T was STR-authenticated by Shanghai Biowing Applied Biotechnology Co. Ltd. NIH3T3, SYF and MEF cells were authenticated as mouse origin by Shanghai Biowing Applied Biotechnology Co. Ltd. All cells were tested for mycoplasma contamination by using GMyc-PCR Mycoplasma Test kit (Yeasen BioTech, Shanghai) and were maintained at 37ºC in a humidified 5% CO2 atmosphere. Antibodies used were anti-STAT3 (cat# sc-8019, sc-482), anti-c-Src (cat# sc-5266), anti-HA, anti-Myc, anti-GFP from Santa Cruz Biotech (Santa Cruz, CA); anti-c-Src (cat# 2109) from Cell Signaling; Anti-Src (phospho Y416) antibody from Abcam (cat# ab185617) and Merck Millipore (cat# 04-857); anti-Na+/K+ ATPase (cat# ab167390), anti-IkB (cat# ab7547), anti-H3 (cat# ab1791), anti-pan acetylated lysine (cat# 22550) from Abcam; anti-GST, and anti-FLAG from Sigma-Aldrich (St. Louis, MO); 4G10 from Millipore (Billerica, MA); anti-pan acetylated lysine (cat# 9441) and anti-pY705-STAT3 (cat# 9145) from Cell Signaling (Boston, MA). Rabbit polyclonal antibodies against acetyl-Src (aK7, aK9, aK423, and aK427) and phospho-STAT3 (pY45 and pY68) were custom prepared by Millipore or AB-land, Inc. (Hangzhou, China). Polyclonal antibodies are produced by immunizing animals with synthetic Downloaded from cancerres.aacrjournals.org on September5 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. acetyl peptides corresponding to residues surrounding acetylated K7, acetylated K9, acetylated K423, or acetylated K427 of Src and synthetic phosphopeptides corresponding to residues surrounding Y45 or Y68 of Stat3. Antibodies were purified by protein A and peptide affinity chromatography. GST or GST tagged Src protein was purchased from Abcam. Recombinant human EGF was purchased from Sigma-Aldrich; recombinant mouse EGF, human OSM, and mouse OSM were purchased from R&D Systems (Minneapolis, MN). For convenience reason, chicken c-Src protein sequence was used in the context, i.e., K401, K423, K427, Y416 and Y527 should be K404, K426, K430, Y419, and Y530 of human Src correspondingly. Preparation
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