Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion. 1. Introduction Glioblastoma multiforme (GBM) is the most common form of all primary adult brain tumors. Although significant technical advances in surgical and radiation treatments for brain tumors have emerged, their impact on clinical outcome for patients has been modest [1, 2]. Of the features that characterize GBM, arguably none is more clinically significant than the propensity of glioma cells to aggressively invade the surrounding normal brain tissue [3]. These invasive cells render complete resection impossible, confer strong resistance to chemo- and radiation therapy, and virtually assure the rise of secondary tumors that develop at the resection margins that drive further invasion [4]. Meaningful advances in clinical outcomes will require identification and targeting of key signaling effectors mediating glioma invasion. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) serves as a point of integration for signaling from cell surface receptors including integrin adhesion receptors, G-protein coupled receptors, and receptor tyrosine kinases [5–7]. As such, signaling from Pyk2 has been implicated in a variety of cellular processes including migration, cell survival, and proliferation. We have demonstrated in glioblastoma tumor samples that Pyk2 expression is upregulated in invasive glioma cells relative to cells in their cognate tumor cores [8] and that increased Pyk2
References
[1]
A. Giese, R. Bjerkvig, M. E. Berens, and M. Westphal, “Cost of migration: invasion of malignant gliomas and implications for treatment,” Journal of Clinical Oncology, vol. 21, no. 8, pp. 1624–1636, 2003.
[2]
R. Stupp, W. P. Mason, M. J. van den Bent et al., “Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma,” The New England Journal of Medicine, vol. 352, no. 10, pp. 987–996, 2005.
[3]
M. E. Berens and A. Giese, “‘...those left behind.’ Biology and oncology of invasive glioma cells,” Neoplasia, vol. 1, no. 3, pp. 208–219, 1999.
[4]
F. Lefranc, J. Brotchi, and R. Kiss, “Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis,” Journal of Clinical Oncology, vol. 23, no. 10, pp. 2411–2422, 2005.
[5]
M. D. Schaller, “Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions,” Journal of Cell Science, vol. 123, no. 7, pp. 1007–1013, 2010.
[6]
I. H. Gelman, “Pyk2 FAKs, any two FAKs,” Cell Biology International, vol. 27, no. 7, pp. 507–510, 2003.
[7]
H. Avraham, S.-Y. Park, K. Schinkmann, and S. Avraham, “RAFTK/Pyk2-mediated cellular signalling,” Cellular Signalling, vol. 12, no. 3, pp. 123–133, 2000.
[8]
D. B. Hoelzinger, L. Mariani, J. Wies et al., “Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets,” Neoplasia, vol. 7, no. 1, pp. 7–16, 2005.
[9]
C. A. Lipinski, N. L. Tran, E. Menashi et al., “The tyrosine kinase Pyk2 promotes migration and invasion of glioma cells,” Neoplasia, vol. 7, no. 5, pp. 435–445, 2005.
[10]
C. A. Lipinski, N. L. Tran, C. Viso et al., “Extended survival of Pyk2 or FAK deficient orthotopic glioma xenografts,” Journal of Neuro-Oncology, vol. 90, no. 2, pp. 181–189, 2008.
[11]
C. A. Lipinski, N. L. Tran, A. Dooley et al., “Critical role of the FERM domain in Pyk2 stimulated glioma cell migration,” Biochemical and Biophysical Research Communications, vol. 349, no. 3, pp. 939–947, 2006.
[12]
J. C. Loftus, Z. Yang, N. L. Tran et al., “The Pyk2 FERM domain as a target to inhibit glioma migration,” Molecular Cancer Therapeutics, vol. 8, no. 6, pp. 1505–1514, 2009.
[13]
A. Astier, H. Avraham, S. N. Manie et al., “The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after β1-integrin stimulation in B cells and binds to p130cas,” Journal of Biological Chemistry, vol. 272, no. 1, pp. 228–232, 1997.
[14]
K. Ueki, T. Mimura, T. Nakamoto et al., “Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK,” FEBS Letters, vol. 432, no. 3, pp. 197–201, 1998.
[15]
S. Lev, H. Moreno, R. Martinez et al., “Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions,” Nature, vol. 376, no. 6543, pp. 737–745, 1995.
[16]
G. Tokiwa, I. Dikic, S. Lev, and J. Schlessinger, “Activation of Pyk2 by stress signals and coupling with JNK signaling pathway,” Science, vol. 273, no. 5276, pp. 792–794, 1996.
[17]
I. Hayashi, K. Vuori, and R. C. Liddington, “The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin,” Nature Structural Biology, vol. 9, no. 2, pp. 101–106, 2002.
[18]
G. Liu, C. D. Guibao, and J. Zheng, “Structural insight into the mechanisms of targeting and signaling of focal adhesion kinase,” Molecular and Cellular Biology, vol. 22, no. 8, pp. 2751–2760, 2002.
[19]
A. Richardson and T. Parsons, “A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK,” Nature, vol. 380, pp. 538–540, 1996.
[20]
A. Richardson, R. K. Malik, J. D. Hildebrand, and J. T. Parsons, “Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation,” Molecular and Cellular Biology, vol. 17, no. 12, pp. 6906–6914, 1997.
[21]
J. Lulo, S. Yuzawa, and J. Schlessinger, “Crystal structures of free and ligand-bound focal adhesion targeting domain of Pyk2,” Biochemical and Biophysical Research Communications, vol. 383, no. 3, pp. 347–352, 2009.
[22]
D. Riggs, Z. Yang, J. Kloss, and J. C. Loftus, “The Pyk2 FERM regulates Pyk2 complex formation and phosphorylation,” Cellular Signalling, vol. 23, no. 1, pp. 288–296, 2011.
[23]
T. Kohno, E. Matsuda, H. Sasaki, and T. Sasaki, “Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca 2+/calmodulin to FERM F2 α2 helix and thus forming its dimer,” Biochemical Journal, vol. 410, no. 3, pp. 513–523, 2008.
[24]
J. M. de Pereda, K. L. Wegener, E. Santelli et al., “Structural basis for phosphatidylinositol phosphate kinase type I gamma binding to talin at focal adhesions,” Journal of Biological Chemistry, vol. 280, no. 9, pp. 8381–8386, 2005.
[25]
B. García-Alvarez, J. M. de Pereda, D. A. Calderwood et al., “Structural determinants of integrin recognition by talin,” Molecular Cell, vol. 11, no. 1, pp. 49–58, 2003.
[26]
K. Hamada, T. Shimizu, S. Yonemura, S. Tsukita, S. Tsukita, and T. Hakoshima, “Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex,” EMBO Journal, vol. 22, no. 3, pp. 502–514, 2003.
[27]
Y. Takai, K. Kitano, S. Terawaki, R. Maesaki, and T. Hakoshima, “Structural basis of PSGL-1 binding to ERM proteins,” Genes to Cells, vol. 12, no. 12, pp. 1329–1338, 2007.
[28]
Y. Takai, K. Kitano, S. I. Terawaki, R. Maesaki, and T. Hakoshima, “Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins,” Journal of Molecular Biology, vol. 381, no. 3, pp. 634–644, 2008.
[29]
T. Mori, K. Kitano, S.-I. Terawaki, R. Maesaki, Y. Fukami, and T. Hakoshima, “Structural basis for CD44 recognition by ERM proteins,” Journal of Biological Chemistry, vol. 283, no. 43, pp. 29602–29612, 2008.
[30]
S.-I. Terawaki, K. Kitano, and T. Hakoshima, “Structural basis for type II membrane protein binding by ERM proteins revealed by the radixin-neutral endopeptidase 24.11 (NEP) complex,” Journal of Biological Chemistry, vol. 282, no. 27, pp. 19854–19862, 2007.
[31]
D. F. J. Ceccarelli, H. K. Song , F. Poy, M. D. Schaller, and M. J. Eck, “Crystal structure of the FERM domain of focal adhesion kinase,” Journal of Biological Chemistry, vol. 281, no. 1, pp. 252–259, 2006.
[32]
D. Lietha, X. Cai, D. F. J. Ceccarelli, Y. Li, M. D. Schaller, and M. J. Eck, “Structural basis for the autoinhibition of focal adhesion kinase,” Cell, vol. 129, no. 6, pp. 1177–1187, 2007.
[33]
J. M. Dunty, V. Gabarra-Niecko, M. L. King, D. F. J. Ceccarelli, M. J. Eck, and M. D. Schaller, “FERM domain interaction promotes FAK signaling,” Molecular and Cellular Biology, vol. 24, no. 12, pp. 5353–5368, 2004.
[34]
C. A. Lipinski, N. L. Tran, C. Bay et al., “Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in determining glioblastoma migration and proliferation,” Molecular Cancer Research, vol. 1, no. 5, pp. 323–332, 2003.
[35]
J. H. Wright, X. Wang, G. Manning et al., “The STE20 kinase HGK is broadly expressed in human tumor cells and can modulate cellular transformation, invasion, and adhesion,” Molecular and Cellular Biology, vol. 23, no. 6, pp. 2068–2082, 2003.
[36]
K. D. Mack, M. V. Goetz, M. Lin et al., “Functional identification of kinases essential for T-cell activation through a genetic suppression screen,” Immunology Letters, vol. 96, no. 1, pp. 129–145, 2005.
[37]
C. S. Collins, J. Hong, L. Sapinoso et al., “A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 10, pp. 3775–3780, 2006.
[38]
A. Giese, M. D. Rief, M. A. Loo, and M. E. Berens, “Determinants of human astrocytoma migration,” Cancer Research, vol. 54, no. 14, pp. 3897–3904, 1994.
[39]
K. Lamszus, N. O. Schmidt, L. Jin et al., “Scatter factor promotes motility of human glioma and neuromicrovascular endothelial cells,” International Journal of Cancer, vol. 75, pp. 19–28, 1998.
[40]
F. Di Cunto, E. Calautti, J. Hsiao et al., “Citron Rho-interacting kinase, a novel tissue-specific Ser/Thr kinase encompassing the Rho-Rac-binding protein citron,” Journal of Biological Chemistry, vol. 273, no. 45, pp. 29706–29711, 1998.
[41]
Y. Su, J. Han, S. Xu, M. Cobb, and E. Skolnik, “NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain,” EMBO Journal, vol. 16, no. 6, pp. 1279–1290, 1997.
[42]
A. H. Chishti, A. C. Kim, S. M. Marfatia et al., “The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane,” Trends in Biochemical Sciences, vol. 23, no. 8, pp. 281–282, 1998.
[43]
S. Lev, J. Hernandez, R. Martinez, A. Chen, G. Plowman, and J. Schlessinger, “Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdGB) protein,” Molecular and Cellular Biology, vol. 19, no. 3, pp. 2278–2288, 1999.
[44]
S. Abbi, H. Ueda, C. Zheng et al., “Regulation of focal adhesion kinase by a novel protein inhibitor FIP200,” Molecular Biology of the Cell, vol. 13, no. 9, pp. 3178–3191, 2002.
[45]
S.-T. Lim, N. L. G. Miller, J.-O. Nam, X. L. Chen, Y. Lim, and D. D. Schlaepfer, “Pyk2 inhibition of p53 as an adaptive and intrinsic mechanism facilitating cell proliferation and survival,” Journal of Biological Chemistry, vol. 285, no. 3, pp. 1743–1753, 2010.
[46]
P. Madaule, T. Furuyashiki, T. Reid et al., “A novel partner for the GTP-bound forms of rho and rac,” FEBS Letters, vol. 377, no. 2, pp. 243–248, 1995.
[47]
P. Poinat, A. De Arcangelis, S. Sookhareea et al., “A conserved interaction between β1 integrin/PAT-3 and Nck-interacting kinase/MIG-15 that mediates commissural axon navigation in C. elegans,” Current Biology, vol. 12, no. 8, pp. 622–631, 2002.
[48]
Z. Yao, G. Zhou, X. S. Wang et al., “A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway,” Journal of Biological Chemistry, vol. 274, no. 4, pp. 2118–2125, 1999.
[49]
J.-M. Hao, J.-Z. Chen, H.-M. Sui et al., “A five-gene signature as a potential predictor of metastasis and survival in colorectal cancer,” Journal of Pathology, vol. 220, no. 4, pp. 475–489, 2010.
[50]
Y.-C. Su, J. E. Treisman, and E. Y. Skolnik, “The Drosophila Ste20-related kinase misshapen is required for embryonic dorsal closure and acts through a JNK MAPK module on an evolutionarily conserved signaling pathway,” Genes and Development, vol. 12, no. 15, pp. 2371–2380, 1998.
[51]
Y. Xue, X. Wang, Z. Li, N. Gotoh, D. Chapman, and E. Y. Skolnik, “Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK),” Development, vol. 128, no. 9, pp. 1559–1572, 2001.
[52]
D. B. Ramnarain, S. Park, D. Y. Lee et al., “Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in glioma cells,” Cancer Research, vol. 66, no. 2, pp. 867–874, 2006.
[53]
M. Baumgartner, A. L. Sillman, E. M. Blackwood et al., “The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 36, pp. 13391–13396, 2006.
[54]
S. Eden, R. Rohatgi, A. V. Podtelejnikov, M. Mann, and M. W. Kirschner, “Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck,” Nature, vol. 418, no. 6899, pp. 790–793, 2002.
[55]
R. Rohatgi, P. Nollau, H.-Y. Ho, M. W. Kirschner, and B. J. Mayer, “Nck and Phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 Pathway,” Journal of Biological Chemistry, vol. 276, no. 28, pp. 26448–26452, 2001.
[56]
N. Machida, M. Umikawa, K. Takei et al., “Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase,” Journal of Biological Chemistry, vol. 279, no. 16, pp. 15711–15714, 2004.
[57]
C. H. Yoon, M. J. Kim, R. K. Kim et al., “c-Jun N-terminal kinase has a pivotal role in the maintenance of self-renewal and tumorigenicity in glioma stem-like cells,” Oncogene, vol. 31, pp. 4655–4666, 2012.
[58]
A. A. Antonyak, L. C. Kenyon, A. K. Godwin et al., “Elevated JNK activation contributes to the pathogenesis of human brain tumors,” Oncogene, vol. 21, no. 33, pp. 5038–5046, 2002.
[59]
J. Cui, S.-Y. Han, C. Wang et al., “c-Jun NH2-terminal kinase 2α2 promotes the tumorigenicity of human glioblastoma cells,” Cancer Research, vol. 66, no. 20, pp. 10024–10031, 2006.
[60]
S. J. McLeod, A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold, “The rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes,” Journal of Biological Chemistry, vol. 279, no. 13, pp. 12009–12019, 2004.
