Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1

Oncogene volume 38, pages 3569–3584 (2019)Cite this article

Subjects

A Correction to this article was published on 17 September 2019

This article has been updated

Abstract

Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Change history

  • 17 September 2019

    The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.

References

  1. Rana A, Rana B, Mishra R, Sondarva G, Rangasamy V, Das S, et al. Mixed lineage kinase-c-Jun N-terminal kinase Axis: a potential therapeutic target in cancer. Genes & Cancer. 2013;4:334–41.

    Article  Google Scholar 

  2. Gallo KA, Johnson GL. Signalling: mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat Rev Mol Cell Biol. 2002;3:663–72.

    Article  CAS  Google Scholar 

  3. Rana A, Gallo K, Godowski P, Hirai S, Ohno S, Zon L, et al. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. J Biol Chem. 1996;271:19025–8.

    Article  CAS  Google Scholar 

  4. Rangasamy V, Mishra R, Mehrotra S, Sondarva G, Ray RS, Rao A, et al. Estrogen suppresses MLK3-mediated apoptosis sensitivity in ER+breast cancer cells. Cancer Res. 2010;70:1731–40.

    Article  CAS  Google Scholar 

  5. Rangasamy V, Mishra R, Sondarva G, Das S, Lee TH, Bakowska JC, et al. Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function. Proc Natl Acad Sci USA. 2012;109:8149–54.

    Article  CAS  Google Scholar 

  6. Mishra P, Senthivinayagam S, Rangasamy V, Sondarva G, Rana B. Mixed lineage kinase-3/JNK1 axis promotes migration of human gastric cancer cells following gastrin stimulation. Mol Endocrinol. 2010;24:598–607.

    Article  CAS  Google Scholar 

  7. Bodner A, Maroney AC, Finn JP, Ghadge G, Roos R, Miller RJ. Mixed lineage kinase 3 mediates gp120IIIB-induced neurotoxicity. J Neurochem. 2002;82:1424–34.

    Article  CAS  Google Scholar 

  8. Parkinson Study Group PI. Mixed lineage kinase inhibitor CEP-1347 fails to delay disability in early Parkinson disease. Neurology. 2007;69:1480–90.

  9. Das S, Sondarva G, Viswakarma N, Nair RS, Osipo C, Tzivion G, et al. Human epidermal growth factor receptor 2 (HER2) impedes MLK3 kinase activity to support breast cancer cell survival. J Biol Chem. 2015;290:21705–12.

    Article  CAS  Google Scholar 

  10. Rattanasinchai C, Llewellyn BJ, Conrad SE, Gallo KA. MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells. Oncogenesis. 2017;6:e345.

    Article  CAS  Google Scholar 

  11. Lee BN, Elion EA. The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components. Proc Natl Acad Sci USA. 1999;96:12679–84.

    Article  CAS  Google Scholar 

  12. Knaus UG, Bokoch GM. The p21Rac/Cdc42-activated kinases (PAKs). Int J Biochem Cell Biol. 1998;30:857–62.

    Article  CAS  Google Scholar 

  13. Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer. Nat Rev Cancer. 2014;14:13–25.

    Article  CAS  Google Scholar 

  14. Letessier A, Sircoulomb F, Ginestier C, Cervera N, Monville F, Gelsi-Boyer V, et al. Frequency, prognostic impact, and subtype association of 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 amplifications in breast cancers. BMC Cancer. 2006;6:245.

    Article  Google Scholar 

  15. French JD, Ghoussaini M, Edwards SL, Meyer KB, Michailidou K, Ahmed S, et al. Functional variants at the 11q13 risk locus for breast cancer regulate cyclin D1 expression through long-range enhancers. Am J Hum Genet. 2013;92:489–503.

    Article  CAS  Google Scholar 

  16. Smith CM, Ma NS, Nowak NJ, Shows TB, Gerhard DS. A 3-Mb contig from D11S987 to MLK3, a gene-rich region in 11q13. Genome Res. 1997;7:835–42.

    Article  CAS  Google Scholar 

  17. Rider L, Oladimeji P, Diakonova M. PAK1 regulates breast cancer cell invasion through secretion of matrix metalloproteinases in response to prolactin and three-dimensional collagen IV. Mol Endocrinol. 2013;27:1048–64.

    Article  CAS  Google Scholar 

  18. Parrini MC, Lei M, Harrison SC, Mayer BJ. Pak1 kinase homodimers are autoinhibited in trans and dissociated upon activation by Cdc42 and Rac1. Mol Cell. 2002;9:73–83.

    Article  CAS  Google Scholar 

  19. Lu W, Mayer BJ. Mechanism of activation of Pak1 kinase by membrane localization. Oncogene. 1999;18:797–806.

    Article  CAS  Google Scholar 

  20. Sathyanarayana P, Barthwal MK, Kundu CN, Lane ME, Bergmann A, Tzivion G, et al. Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3. Mol Cell. 2002;10:1527–33.

    Article  CAS  Google Scholar 

  21. Nheu TV, He H, Hirokawa Y, Tamaki K, Florin L, Schmitz ML, et al. The K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS transformants. Cancer J. 2002;8:328–36.

    Article  Google Scholar 

  22. Jha RK, Wu YI, Zawistowski JS, MacNevin C, Hahn KM, Kuhlman B. Redesign of the PAK1 autoinhibitory domain for enhanced stability and affinity in biosensor applications. J Mol Biol. 2011;413:513–22.

    Article  CAS  Google Scholar 

  23. Heo L, Lee H, Seok C. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking. Sci Rep. 2016;6:32153.

    Article  CAS  Google Scholar 

  24. Ong CC, Jubb AM, Haverty PM, Zhou W, Tran V, Truong T, et al. Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells. Proc Natl Acad Sci USA. 2011;108:7177–82.

    Article  CAS  Google Scholar 

  25. Frost JA, Swantek JL, Stippec S, Yin MJ, Gaynor R, Cobb MH. Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1. J Biol Chem. 2000;275:19693–9.

    Article  CAS  Google Scholar 

  26. Qing H, Gong W, Che Y, Wang X, Peng L, Liang Y, et al. PAK1-dependent MAPK pathway activation is required for colorectal cancer cell proliferation. Tumour Biol. 2012;33:985–94.

    Article  CAS  Google Scholar 

  27. Wang Z, Fu M, Wang L, Liu J, Li Y, Brakebusch C, et al. p21-activated kinase 1 (PAK1) can promote ERK activation in a kinase-independent manner. J Biol Chem. 2013;288:20093–9.

    Article  CAS  Google Scholar 

  28. Mao K, Kobayashi S, Jaffer ZM, Huang Y, Volden P, Chernoff J, et al. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes. J Mol Cell Cardiol. 2008;44:429–34.

    Article  CAS  Google Scholar 

  29. Kumar R, Vadlamudi RK. Emerging functions of p21-activated kinases in human cancer cells. J Cell Physiol. 2002;193:133–44.

    Article  CAS  Google Scholar 

  30. Misek SA, Chen J, Schroeder L, Rattanasinchai C, Sample A, Sarkaria JN, et al. EGFR signals through a DOCK180-MLK3 axis to drive glioblastoma cell invasion. Mol Cancer Res. 2017;15:1085–95.

    Article  CAS  Google Scholar 

  31. Nayal A, Webb DJ, Brown CM, Schaefer EM, Vicente-Manzanares M, Horwitz AR. Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics. J Cell Biol. 2006;173:587–9.

    Article  CAS  Google Scholar 

  32. Wang RA, Mazumdar A, Vadlamudi RK, Kumar R. P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium. EMBO J. 2002;21:5437–47.

    Article  CAS  Google Scholar 

  33. Chen RE, Thorner J. Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta. 2007;1773:1311–40.

    Article  CAS  Google Scholar 

  34. Chong C, Tan L, Lim L, Manser E. The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity. J Biol Chem. 2001;276:17347–53.

    Article  CAS  Google Scholar 

  35. Mayhew MW, Jeffery ED, Sherman NE, Nelson K, Polefrone JM, Pratt SJ, et al. Identification of phosphorylation sites in betaPIX and PAK1. J Cell Sci. 2007;120:3911–8.

    Article  CAS  Google Scholar 

  36. Holm C, Rayala S, Jirstrom K, Stal O, Kumar R, Landberg G. Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst. 2006;98:671–80.

    Article  CAS  Google Scholar 

  37. Zhou L, Yan C, Gieling RG, Kida Y, Garner W, Li W, et al. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated kinase-1. BMC Immunol. 2009;10:15.

    Article  Google Scholar 

  38. Tabusa H, Brooks T, Massey AJ. Knockdown of PAK4 or PAK1 inhibits the proliferation of mutant KRAS colon cancer cells independently of RAF/MEK/ERK and PI3K/AKT signaling. Mol Cancer Res. 2013;11:109–21.

    Article  CAS  Google Scholar 

  39. Sancho D, Montoya MC, Monjas A, Gordon-Alonso M, Katagiri T, Gil D, et al. TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion. J Immunol. 2002;169:292–300.

    Article  CAS  Google Scholar 

  40. Lu W, Katz S, Gupta R, Mayer BJ. Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck. Curr Biol. 1997;7:85–94.

    Article  CAS  Google Scholar 

  41. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010;5:725–38.

    Article  CAS  Google Scholar 

  42. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods. 2015;12:7–8.

    Article  CAS  Google Scholar 

  43. Zhang C, Mortuza SM, He B, Wang Y, Zhang Y. Template-based and free modeling of I-TASSER and QUARK pipelines using predicted contact maps in CASP12. Proteins. 2018;86 (Suppl 1):136–51.

    Article  CAS  Google Scholar 

  44. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinforma. 2008;9:40.

    Article  Google Scholar 

  45. Yang J, Zhang Y. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 2015;43:W174–81.

    Article  CAS  Google Scholar 

  46. Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics. 2014;30:1771–3.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge funding support from National Cancer Institute to A.R. (CA 176846 and CA 216410), B.R. (CA 178063) and V.G. (CA 188427). Additional support came from Veteran Administration Merit Awards to A.R. (BX002703) and B.R. (BX003296). We also acknowledge the NIH shared instrument grant # S100D018445, awarded to U.A.M.S. Proteomics Core Lab, Little Rock, Arkansas, USA.

Author information

Author notes

  1. These authors contributed equally: Subhasis Das, Rakesh Sathish Nair

Authors and Affiliations

  1. Department of Surgery, Division of Surgical Oncology, University of Illinois at Chicago, Chicago, IL, 60612, USA

    Subhasis Das, Rakesh Sathish Nair, Gautam Sondarva, Navin Viswakarma, Basabi Rana & Ajay Rana

  2. Center for Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, 835205, India

    Rajakishore Mishra

  3. Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, 60607, USA

    Hazem Abdelkarim & Vadim Gaponenko

  4. University of Illinois Hospital &Health Sciences System Cancer Center, University of Illinois at Chicago, Chicago, IL, 60612, USA

    Basabi Rana & Ajay Rana

  5. Jesse Brown VA Medical Center, Chicago, IL, 60612, USA

    Basabi Rana & Ajay Rana

Authors
  1. Subhasis Das
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rakesh Sathish Nair
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rajakishore Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gautam Sondarva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Navin Viswakarma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hazem Abdelkarim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vadim Gaponenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Basabi Rana
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ajay Rana
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ajay Rana.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Das, S., Nair, R.S., Mishra, R. et al. Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1. Oncogene 38, 3569–3584 (2019). https://doi.org/10.1038/s41388-019-0690-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-019-0690-0

This article is cited by

Search

Advanced search

Quick links