Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Background: Rac 1 GTPase mediates glycogen phosphorylase activation and controls IL-2-stimulated T cell proliferation. Results: PKCθ activates αPIX by serine phosphorylation and now this Rho-GEF activates Rac 1. Conclusion: IL-2-stimulated T cells migration and proliferation require the involvement of the PKCθ/αPIX/Rac 1/PYGM pathway. Significance: This new signaling cascade may be a viable therapeutic target to block the inflammatory response mediated by T cells. Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation.

[1]  M. Moses,et al.  PKCθ Regulates T Cell Motility via Ezrin-Radixin-Moesin Localization to the Uropod , 2013, PloS one.

[2]  Yong Ryoul Yang,et al.  The physiological roles of primary phospholipase C. , 2013, Advances in biological regulation.

[3]  C. Geisler,et al.  PKC‐θ exists in an oxidized inactive form in naive human T cells , 2013, European journal of immunology.

[4]  Takeshi Tomonaga,et al.  Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project. , 2013, Journal of proteome research.

[5]  J. Zugaza,et al.  1–42 β-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death , 2013, Translational Psychiatry.

[6]  A. Altman,et al.  PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors , 2012, Front. Immun..

[7]  A. Long,et al.  Regulating the Regulator: Phosphorylation of PKC θ in T Cells , 2012, Front. Immun..

[8]  T. Tan,et al.  Regulation of PKC-θ function by phosphorylation in T cell receptor signaling , 2012, Front. Immun..

[9]  J. Zugaza,et al.  Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation* , 2012, The Journal of Biological Chemistry.

[10]  H. Daub,et al.  Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells. , 2012, Journal of proteomics.

[11]  B. Blagoev,et al.  Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics. , 2011, Journal of proteomics.

[12]  Jennifer M. Bolin,et al.  Proteomic and phosphoproteomic comparison of human ES and iPS cells , 2011, Nature Methods.

[13]  M. Hoshino,et al.  Phosphorylation of STEF/Tiam2 by protein kinase A is critical for Rac1 activation and neurite outgrowth in dibutyryl cAMP–treated PC12D cells , 2011, Molecular biology of the cell.

[14]  Paola Chiarugi,et al.  Rac and Rho GTPases in cancer cell motility control , 2010, Cell Communication and Signaling.

[15]  T. Malek,et al.  Interleukin-2 receptor signaling: at the interface between tolerance and immunity. , 2010, Immunity.

[16]  Hans Janssen,et al.  The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration. , 2009, Blood.

[17]  X. Bustelo,et al.  Mechanistic Analysis of the Amplification and Diversification Events Induced by Vav Proteins in B-lymphocytes* , 2008, Journal of Biological Chemistry.

[18]  G. Obermair,et al.  PKC-theta selectively controls the adhesion-stimulating molecule Rap1. , 2008, Blood.

[19]  C. Selmi,et al.  The regulatory, inflammatory, and T cell programming roles of interleukin-2 (IL-2). , 2008, Journal of autoimmunity.

[20]  Jun O. Liu,et al.  Nuclear Export of NF90 to Stabilize IL-2 mRNA Is Mediated by AKT-Dependent Phosphorylation at Ser647 in Response to CD28 Costimulation1 , 2008, The Journal of Immunology.

[21]  M. Hoshino,et al.  Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing , 2007, FEBS letters.

[22]  X. Bustelo,et al.  GTP‐binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo , 2007, BioEssays : news and reviews in molecular, cellular and developmental biology.

[23]  D. Billadeau,et al.  Regulation of T-cell activation by the cytoskeleton , 2007, Nature Reviews Immunology.

[24]  C. Pike,et al.  Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen , 2006, Journal of neurochemistry.

[25]  M. Neurath,et al.  Azathioprine Suppresses Ezrin-Radixin-Moesin-Dependent T Cell-APC Conjugation through Inhibition of Vav Guanosine Exchange Activity on Rac Proteins , 2006, The Journal of Immunology.

[26]  A. Wells,et al.  Signals from CD28 Induce Stable Epigenetic Modification of the IL-2 Promoter1 , 2005, The Journal of Immunology.

[27]  E. Arias-Palomo,et al.  Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3 , 2005, The EMBO journal.

[28]  O. Bernard,et al.  Interaction of the tyrosine phosphatase SHP-2 with Gab2 regulates Rho-dependent activation of the c-fos serum response element by interleukin-2. , 2004, The Biochemical journal.

[29]  Q. Feng,et al.  Novel regulatory mechanisms for the Dbl family guanine nucleotide exchange factor Cool‐2/α‐Pix , 2004 .

[30]  X. Bustelo,et al.  Inverted signaling hierarchy between RAS and RAC in T-lymphocytes , 2004, Oncogene.

[31]  G. Bokoch.  Biology of the p21-activated kinases. , 2003, Annual review of biochemistry.

[32]  X. Bustelo,et al.  Exchange Factors of the RasGRP Family Mediate Ras Activation in the Golgi* , 2003, Journal of Biological Chemistry.

[33]  D. Matallanas,et al.  Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1 , 2003, The EMBO journal.

[34]  S. Meloche,et al.  Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors , 2003, Molecular and Cellular Biology.

[35]  N. Westergaard,et al.  The effect of glucose on the potency of two distinct glycogen phosphorylase inhibitors. , 2002, The Biochemical journal.

[36]  D. Littman,et al.  Protein kinase C-θ: signaling from the center of the T-cell synapse , 2002 .

[37]  W. Pawlina,et al.  Glycogen phosphorylase is activated in response to glucose deprivation but is not responsible for enhanced glucose transport activity in 3T3-L1 adipocytes. , 2002, Biochimica et biophysica acta.

[38]  M. Hoshino,et al.  Characterization of STEF, a Guanine Nucleotide Exchange Factor for Rac1, Required for Neurite Growth* , 2002, The Journal of Biological Chemistry.

[39]  Y. Nishizuka.  The protein kinase C family and lipid mediators for transmembrane signaling and cell regulation. , 2001, Alcoholism, clinical and experimental research.

[40]  T. Mustelin,et al.  Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway. , 2000, European cytokine network.

[41]  J. Bertoglio,et al.  IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes , 2000, Oncogene.

[42]  D. Green,et al.  Protein kinase ctheta cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death. , 1999, Journal of immunology.

[43]  H. Sugimura,et al.  αPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase , 1999, Oncogene.

[44]  C. Hall,et al.  PAK promotes morphological changes by acting upstream of Rac , 1998, The EMBO journal.

[45]  A. Trautmann,et al.  Involvement of phosphoinositide 3‐kinase and Rac in membrane ruffling induced by IL‐2 in T cells , 1998, European journal of immunology.

[46]  John G. Collard,et al.  Lysophosphatidic Acid Induces Threonine Phosphorylation of Tiam1 in Swiss 3T3 Fibroblasts via Activation of Protein Kinase C* , 1997, The Journal of Biological Chemistry.

[47]  C. Martínez-A,et al.  IL-2 signaling controls actin organization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta. , 1997, Journal of immunology.

[48]  K. Schuebel,et al.  Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product , 1997, Nature.

[49]  S. Fesik,et al.  Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[50]  H. Grunicke,et al.  Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes , 1996, Molecular and cellular biology.

[51]  S. Shoelson,et al.  Interaction of Shc with Grb2 Regulates the Grb2 Association with mSOS , 1995, Annals of the New York Academy of Sciences.

[52]  Y. Nishizuka.  Protein kinase C and lipid signaling for sustained cellular responses , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[53]  F. Marks,et al.  Rottlerin, a novel protein kinase inhibitor. , 1994, Biochemical and biophysical research communications.

[54]  W. Farrar,et al.  Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck. , 1993, The Biochemical journal.

[55]  H. Ohno,et al.  Establishment of an interleukin 2-dependent human T cell line from a patient with T cell chronic lymphocytic leukemia who is not infected with human T cell leukemia/lymphoma virus. , 1987, Blood.

[56]  Jacqueline Cherfils,et al.  Regulation of small GTPases by GEFs, GAPs, and GDIs. , 2013, Physiological reviews.

[57]  P. Crespo,et al.  Activation of Ras and Rho GTPases and MAP Kinases by G-protein-coupled receptors. , 2010, Methods in molecular biology.

[58]  H. Vetter,et al.  Epidermal growth factor stimulates Rac1 and p21-activated kinase in vascular smooth muscle cells. , 2008, Atherosclerosis.

[59]  G. Obermair,et al.  PKC-selectively controls the adhesion-stimulating molecule Rap 1 , 2008 .

[60]  Sun,et al.  Selective Function of PKC-θ in T cells , 2006 .

[61]  S. Manicassamy,et al.  Selective function of PKC-theta in T cells. , 2006, Cellular & molecular immunology.

[62]  K. Jakobs,et al.  Mammalian phospholipase C , 2003 .

[63]  X. Q. Chen,et al.  PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. , 1998, Molecular cell.

[64]  John G. Collard,et al.  Role of Tiam 1 in Rac-mediated signal transduction pathways. , 1996, Current topics in microbiology and immunology.

[65]  K. Smith.  The bimolecular structure of the interleukin 2 receptor. , 1988, Immunology today.