Porcine ARAF Interaction with MST2 Regulated Apoptosis in PK-15 Cells
DOI:
https://doi.org/10.22456/1679-9216.152983Keywords:
ARAF, MST2, Protein–protein interaction, ApoptosisAbstract
Background: Apoptosis is a conserved cellular process essential for tissue homeostasis and host defense regulation. ARAF and MST2 are key serine/threonine kinases that regulate cell survival and apoptosis. However, whether these proteins directly interact and how their association affects apoptotic signaling remain unclear. In human cells, ARAF–MST2 interaction regulates apoptosis independently of the canonical Raf/MEK/ERK pathway. Therefore, this study aimed to determine whether porcine ARAF interacts with MST2 and to elucidate the molecular mechanism by which this interaction influences apoptosis in PK-15 cells.
Materials, Methods & Results: Coding sequences of porcine ARAF and MST2 genes were amplified, cloned into pcDNA3.1 expression vectors with N-terminal Flag and HA epitope tags, verified by Sanger sequencing, and transiently transfected into PK-15 cells. Bioinformatic analyses using STRING and AlphaFold3 predicted a strong physical association between ARAF and MST2, with the predicted interaction interface involving several conserved residues within α-helical and random-coil domains. Structural visualization through PyMOL and secondary structure prediction by SOPMA and SWISS-MODEL revealed highly conserved domains consistent with functional interaction potential. Coimmunoprecipitation assays verified the formation of the ARAF–MST2 complex in PK-15 cells, and immunofluorescence microscopy demonstrated that both proteins predominantly co-localized in the cytoplasm, in agreement with UniProt-based localization predictions. Functional assays revealed that ARAF overexpression markedly reduced PK-15 cell apoptosis, as determined by Annexin V/PI flow cytometric analysis. Western blot and quantitative PCR analyses further showed that ARAF overexpression led to significant suppression of MST2 phosphorylation at threonine 180 (p-MST2 T180), accompanied by increased total MST2 and BCL-2 levels, and decreased pro-apoptotic BAX and cleaved caspase-3 (Cl-caspase3) expression. Conversely, when MST2 activation was enhanced, apoptotic signaling was restored, indicating that ARAF modulates cell fate through inhibition of MST2 activation. Statistical analyses, conducted via one-way ANOVA and t-tests across three biological replicates.
Discussion: Collectively, these findings establish that porcine ARAF physically interacts with MST2 and exerts an antiapoptotic effect by attenuating MST2 phosphorylation and activation. The discovery of the ARAF–MST2 axis as a negative regulator of apoptotic signaling expands our understanding of how Raf kinases integrate with Hippo pathway components to balance cellular survival and death. Given the evolutionary conservation of ARAF and MST2 across mammalian species, this study provides a new molecular framework for exploring how ARAF–MST2 crosstalk contributes to host–pathogen interactions, cellular stress responses, and the maintenance of homeostasis in porcine cells. Furthermore, the elucidation of this novel ARAF–MST2 signaling mechanism lays the groundwork for future research into its role in viral infections, oncogenic transformation, and other apoptosis-related diseases in swine and potentially in other mammals. Overall, our data indicate that the ARAF–MST2 complex functions as a pivotal modulator of cell fate by integrating pro- and anti-apoptotic cues within the cytoplasmic signaling network of PK-15 cells.
Keywords: ARAF, MST2, protein–protein interaction, apoptosis.
Downloads
References
1 Ardestani A., Lupse B. & Maedler K. 2018. Hippo Signaling: Key Emerging Pathway in Cellular and Whole-Body Metabolism. Trends in Endocrinology and Metabolism: TEM. 29(7): 492-509.DOI:10.1016/j.tem.2018.04.006.
2 Avruch J., Zhou D., Fitamant J., Bardeesy N., Mou F. & Barrufet L.R. 2012. Protein kinases of the Hippo pathway: regulation and substrates. Seminars in Cell & Developmental Biology. 23(7): 770-784. DOI: 10.1016/j.semcdb.2012.07.002.
3 Beazely M.A., Alan J.K. & Watts V.J. 2005. Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells. Molecular Pharmacology. 67(1): 250-259. DOI:10.1124/mol.104.001370.
4 Bonner T.I., Kerby S.B., Sutrave P., Gunnell M.A., Mark G. & Rapp U.R. 1985. Structure and biological activity of human homologs of the raf/mil oncogene. Molecular and Cellular Biology. 5(6): 1400-1407. DOI:10.1128/mcb.5.6.1400-1407.1985.
5 Cairns L., Tran T. & Kavran J.M. 2017. Structural Insights into the Regulation of Hippo Signaling. ACS Chemical Biology. 12(3): 601-610. DOI:10.1021/acschembio.6b01058.
6 Christofk H.R., Vander Heiden M.G., Harris M.H., Ramanathan A., Gerszten R.E., Wei R., Fleming M.D.,
Schreiber S.L. & Cantley L.C. 2008. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 452(7184): 230-233. DOI:10.1038/nature06734.
7 Davies H., Bignell G.R., Cox C., Stephens P., Edkins S., Clegg S., Teague J., Woffendin H., Garnett M.J., Bottomley W., Davis N., Dicks E., Ewing R., Floyd Y., Gray K., Hall S., Hawes R., Hughes J., Kosmidou V., MenziesA., Mould C., Parker A., Stevens C., Watt S., Hooper S., Wilson R., Jayatilake H., Gusterson B.A., Cooper C., Shipley J., Hargrave D., Pritchard-Jones K., Maitland N., Chenevix-Trench G., Riggins G.J., Bigner D.D., Palmieri G., Cossu A., Flanagan A., Nicholson A., Ho J.W.C., Leung S.Y., Yuen S.T., Weber B.L., Seigler H.F., Darrow T.L., Paterson H., Marais R., Marshall C.J., Wooster R., Stratton M.R. & Futreal P.A. 2002. Mutations of the BRAF gene in human cancer. Nature. 417(6892): 949-954. DOI:10.1111/ced.12015
8 Du X., Dong Y., Shi H., Li J., Kong S., Shi D., Sun L.V., Xu T., Deng K. & Tao W. 2014. Mst1 and mst2 are essential regulators of trophoblast differentiation and placenta morphogenesis. PloS One. 9(3): e90701. DOI: 10.1371/journal.pone.0090701.
9 Han M., Golden A., Han Y. & Sternberg P.W. 1993. C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation. Nature. 363(6425): 133-140. DOI:10.1038/363133a0.
10 Karasarides M., Chiloeches A., Hayward R., Niculescu-Duvaz D., Scanlon I., Friedlos F., Ogilvie L., Hedley D., Martin J., Marshall C.J., Springer C.J. & Marais R. 2004. B-RAF is a therapeutic target in melanoma. Oncogene. 23(37): 6292-6298. DOI:10.1038/sj.onc.1207785.
11 Khan P.S., Rajesh P., Rajendra P., Chaskar M.G., Rohidas A. & Jaiprakash S. 2022. Recent advances in B-RAF inhibitors as anticancer agents. Bioorganic Chemistry. 120: 105597. DOI:10.1016/.bioorg.2022.105597.
12 Liu S., Su D., Sun Z., Guan L., Wang Z., Zhang G., Zheng G., Cui T., Ma X. & Hu S. 2022. High MST2 expression regulates lens epithelial cell apoptosis in age-related cataracts through YAP1 targeting GLUT1. Archives of Biochemistry and Biophysics. 723: 109255. DOI:10.1016/j.abb.2022.109255.
13 Livak K.J. & Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25(4): 402-408. DOI:10.1006/meth.2001.1262.
14 Marais R., Light Y., Paterson H.F., Mason C.S. & Marshall C.J. 1997. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. The Journal of Biological Chemistry. 272(7): 4378-4383. DOI:10.1074/jbc.272.7.4378.
15 Masliantsev K., Karayan-Tapon L. & Guichet P.-O. 2021. Hippo Signaling Pathway in Gliomas. Cells. 10(1): 184. DOI:10.3390/cells10010184.
16 Matallanas D., Birtwistle M., Romano D., Zebisch A., Rauch J., von Kriegsheim A. & Kolch W. 2011. Raf family kinases: old dogs have learned new tricks. Genes & Cancer. 2(3): 232-260. DOI: 10.1177/1947601911407323.
17 Matallanas D., Romano D., Yee K., Meissl K., Kucerova L., Piazzolla D., Baccarini M., Vass J.K., Kolch W. &
O’neill E. 2007. RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Molecular Cell. 27(6): 962-975. DOI:10.1016/j.molcel.2007.08.008.
18 Mazurek S., Drexler H.C.A., Troppmair J., Eigenbrodt E. & Rapp U.R. 2007. Regulation of pyruvate kinase type M2 by A-Raf: a possible glycolytic stop or go mechanism. Anticancer Research. 27(6B): 3963-3971.PMID: 18225557.
19 McCubrey J.A., Steelman L.S., Hoyle P.E., Blalock W.L., Weinstein-Oppenheimer C., Franklin R.A., Cherwinski H., Bosch E. & McMahon M. 1998. Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. Leukemia. 12(12):1903-1929. DOI:10.1038/sj.leu.2401215.
20 Moodie S.A., Willumsen B.M., Weber M.J. & Wolfman A. 1993. Complexes of Ras.GTP with Raf-1 and mitogenactivated protein kinase kinase. Science. 260(5114): 1658-1661. DOI:10.1126/science.8503013.
21 Odashima M., Usui S., Takagi H., Hong C., Liu J., Yokota M. & Sadoshima J. 2007. Inhibition of endogenous Mst1 prevents apoptosis and cardiac dysfunction without affecting cardiac hypertrophy after myocardial infarction. Circulation Research. 100(9): 1344-1352. DOI:10.1161/01.RES.0000265846.23485.7a.
22 Oh S., Lee D., Kim T., Kim T.-S., Oh H.J., Hwang C.Y., Kong Y.Y., Kwon K.S. & Lim D.S. 2009. Crucial role for Mst1 and Mst2 kinases in early embryonic development of the mouse. Molecular and Cellular Biology. 29(23): 6309-6320. DOI:10.1128/MCB.00551-09.
23 O’Neill E. & Kolch W. 2005. Taming the Hippo: Raf-1 controls apoptosis by suppressing MST2/Hippo. Cell Cycle (Georgetown, Tex.). 4(3): 365-367. DOI:10.4161/cc.4.3.1531.
24 O’Neill E., Rushworth L., Baccarini M. & Kolch W. 2004. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science. 306(5705): 2267-2270. DOI:10.1126/science.1103233.
25 Pritchard C.A., Samuels M.L., Bosch E. & McMahon M. 1995. Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. Molecular and Cellular Biology. 15(11): 6430-6442. DOI:10.1128/MCB.15.11.6430.
26 Rapp U.R., Goldsborough M.D., Mark G.E., Bonner T.I., Groffen J., Reynolds F.H. & Stephenson J.R. 1983. Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus. Proceedings of the National Academy of Sciences of the United States of America. 80(14): 4218-4222. DOI: 10.1073/pnas.80.14.4218.
27 Rauch J., O’Neill E., Mack B., Matthias C., Munz M., Kolch W. & Gires O. 2010. Heterogeneous nuclear ribonu-
cleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription. Cancer Research. 70(4): 1679-1688. DOI: 10.1158/0008-5472.CAN-09-2740.
28 Rauch J., Vandamme D., Mack B., McCann B., Volinsky N., Blanco A., Gires O. & Kolch W. 2016. Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation. Cell Death and Differentiation. 23(8): 1283-1295. DOI:10.1038/cdd.2016.2.
29 Rawat S.J. & Chernoff J. 2015. Regulation of mammalian Ste20 (Mst) kinases. Trends in Biochemical Sciences. 40(3): 149-156. DOI:10.1016/j.tibs.2015.01.001.
30 Romano D., Nguyen L.K., Matallanas D., Halasz M., Doherty C., Kholodenko B.N. & Kolch W. 2014. Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling. Nature Cell Biology. 16(7): 673-684. DOI:10.1038/
ncb2986.
31 Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D., Amin N., Schwikowski B. & Ideker T. 2003. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research. 13(11): 2498-2504. DOI:10.1101/gr.1239303.
32 Sharma J., Antenos M. & Madan P. 2021. A Comparative Analysis of Hippo Signaling Pathway Components during Murine and Bovine Early Mammalian Embryogenesis. Genes. 12(2): 281. DOI:10.3390/genes12020281.
33 Szklarczyk D., Morris J.H., Cook H., Kuhn M., Wyder S., Simonovic M., Santos A., Doncheva N.T., Roth A., Bork P., Jensen L.J. & von Mering C. 2017. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Research. 45(D1): D362-D368. DOI:10.1093/nar/gkw937.
34 Won G.W., Park S.H., Park J., Lee Y. & Lee Y.H. 2019. Mammalian Hippo kinase pathway is downregulated by BCL-2 via protein degradation. Biochemical and Biophysical Research Communications. 512(1): 87-92. DOI:10.1016/j.bbrc.2019.03.015.
35 Xu K., Zhu S., Xu F., Yang J., Deng B., Su D., Ma J., Zu M., Lin Y., Pei T., Zhu Y., Wang L., Liu D., Duan Q.,
Xu J., Pan Z., Tao J. & Hou Z. 2024. Toxoplasma gondii induces MST2 phosphorylation mediating the activation of hippo signaling pathway to promote apoptosis and lung tissue damage. iScience. 27(12): 111312. DOI: 10.1016/j.isci.2024.111312.
36 Yamamoto S., Yang G., Zablocki D., Liu J., Hong C., Kim S.J., Soler S., Odashima M., Thaisz J., Yehia G., Molina C.A., Yatani A., Vatner D.E., Vatner S.F. & Sadoshima J. 2003. Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy. The Journal of Clinical Investigation. 111(10): 1463-1474. DOI:10.1172/JCI17459.
37 Yoon S. & Seger R. 2006. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors. 24(1): 21-44. DOI:10.1080/02699050500284218.
38 Yu F.-X., Zhao B. & Guan K.-L. 2015. Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer. Cell. 163(4): 811-828. DOI:10.1016/j.cell.2015.10.044.
39 Zhang J., Liu J., Gao S., Lin W., Gao P., Gao K., Zhang Y., Du K., Yang X., Wang W., Zhu R. & Wang Y. 2019. Antiapoptosis and Antifibrosis Effects of Qishen Granules on Heart Failure Rats via Hippo Pathway. BioMed Research International. 2019: 1642575. DOI: 10.1155/2019/1642575.
40 Zhang Q., Cheng X., Zhang H., Zhang T., Wang Z., Zhang W. & Yu W. 2020. Dissecting molecular mechanisms underlying H2O2-induced apoptosis of mouse bone marrow mesenchymal stem cell: role of Mst1 inhibition. Stem Cell Research & Therapy. 11(1): 526. DOI:10.1186/s13287-020-02041-7.
41 Zhang X.F., Settleman J., Kyriakis J.M., Takeuchi-Suzuki E., Elledge S.J., Marshall M.S., Bruder J.T., Rapp U.R. & Avruch J. 1993. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 364(6435): 308-313. DOI:10.1038/364308a0.
42 Zheng Y. & Pan D. 2019. The Hippo Signaling Pathway in Development and Disease. Developmental Cell. 50(3): 264-282. DOI:10.1016/j.devcel.2019.06.003.
Additional Files
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Linwei Dai, Kangzhi Xu, Zhiyong Han, Jiexin Yin, Beibei Li, Jing Ma, Mingyue Zu, Jianping Tao, Zhaofeng Hou
This work is licensed under a Creative Commons Attribution 4.0 International License.
This journal provides open access to all of its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Such access is associated with increased readership and increased citation of an author's work. For more information on this approach, see the Public Knowledge Project and Directory of Open Access Journals.
We define open access journals as journals that use a funding model that does not charge readers or their institutions for access. From the BOAI definition of "open access" we take the right of users to "read, download, copy, distribute, print, search, or link to the full texts of these articles" as mandatory for a journal to be included in the directory.
La Red y Portal Iberoamericano de Revistas Científicas de Veterinaria de Libre Acceso reúne a las principales publicaciones científicas editadas en España, Portugal, Latino América y otros países del ámbito latino
