Constitutive Activity of JNK2α2 Is Dependent on a Unique Mechanism of MAPK Activation

Abstract

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2α2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2α2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2α2 autophosphorylation and autoactivation. We find that JNK2α2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the α-region. Alanine scanning mutagenesis of the α-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2α2 dimerization rendering JNK2α2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2α2 homodimerization. We also show that a kinase-inactive JNK2α2 mutant can interact with and inhibit wild type JNK2α2 autophosphorylation, suggesting that JNK2α2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2α2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2α2 activity and that prevention of dimerization may decrease JNK2α2 induced tumorigenic phenotypes. c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2α2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2α2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2α2 autophosphorylation and autoactivation. We find that JNK2α2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the α-region. Alanine scanning mutagenesis of the α-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2α2 dimerization rendering JNK2α2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2α2 homodimerization. We also show that a kinase-inactive JNK2α2 mutant can interact with and inhibit wild type JNK2α2 autophosphorylation, suggesting that JNK2α2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2α2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2α2 activity and that prevention of dimerization may decrease JNK2α2 induced tumorigenic phenotypes. The c-Jun N-terminal kinases (JNK), 3The abbreviations used are: JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; WT, wild type; YFP, yellow fluorescent protein; BS3, bis(sulfosuccinimidyl) suberate. along with ERK1/2 and p38, comprise the mitogen-activated protein kinase (MAPK) family. By regulating AP-1 complexes and other transcription factors, JNK can regulate apoptosis, cellular proliferation, and differentiation (1.Kyriakis J.M. Liu H. Chadee D.N. Methods Mol. Biol. 2004; 250: 61-88PubMed Google Scholar, 2.Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3616) Google Scholar). Members of the JNK family have emerged as having important and diverse functions, depending on the isoform. Three distinct genes encoding JNKs are known: JNK1, JNK2, and JNK3, and at least 10 different splicing isoforms exist in mammalian cells. JNK1 and JNK2 are expressed in all tissues, whereas JNK3 expression is restricted to the brain, heart, and testis (3.Bode A.M. Dong Z. Mol. Carcinog. 2007; 46: 591-598Crossref PubMed Scopus (226) Google Scholar). JNK1 and JNK2 were initially believed to possess redundant functions; however, it was found that JNK1 preferentially mediates apoptosis (4.Butterfield L. Storey B. Maas L. Heasley L.E. J. Biol. Chem. 1997; 272: 10110-10116Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), whereas JNK2 is associated with cellular proliferation (5.Arbour N. Naniche D. Homann D. Davis R.J. Flavell R.A. Oldstone M.B. J. Exp. Med. 2002; 195: 801-810Crossref PubMed Scopus (77) Google Scholar). These findings suggest that individual JNK isoforms may play different roles in cellular transformation. Recently, JNK2 has been implicated in tumorigenesis because JNK2-deficient mice were more resistant to the induction of papillomas, and silencing of JNK2 through RNA interference was shown to hinder the growth of the human T98G glioblastoma cell line (6.Chen N. Nomura M. She Q.B. Ma W.Y. Bode A.M. Wang L. Flavell R.A. Dong Z. Cancer Res. 2001; 61: 3908-3912PubMed Google Scholar, 7.Potapova O. Gorospe M. Bost F. Dean N.M. Gaarde W.A. Mercola D. Holbrook N.J. J. Biol. Chem. 2000; 275: 24767-24775Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We have also previously shown that 86% of primary glial tumors have increased activation of a specific JNK2 isoform, JNK2α2 (8.Tsuiki H. Tnani M. Okamoto I. Kenyon L.C. Emlet D.R. Holgado-Madruga M. Lanham I.S. Joynes C.J. Vo K.T. Wong A.J. Cancer Res. 2003; 63: 250-255PubMed Google Scholar, 9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google Scholar). In addition, we demonstrated that overexpression of JNK2α2 increased cell growth and increased tumor formation in nude mice, indicating that JNK2α2 may be important for tumor formation (9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google Scholar). JNK2α2 was also found to activate two crucial effectors of tumorigenesis: increased phosphorylation of AKT and increased eIF4E levels. AKT is a critical mediator for cell survival and proliferation, and eIF4E is a component of a cap-dependent translation initiation complex that is linked to breast, colon, and prostate tumors (9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google Scholar, 10.Avdulov S. Li S. Michalek V. Burrichter D. Peterson M. Perlman D.M. Manivel J.C. Sonenberg N. Yee D. Bitterman P.B. Polunovsky V.A. Cancer Cell. 2004; 5: 553-563Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Previous work argues that MAPK proteins, such as ERK1/2 and JNK, can dimerize and that dimerization may play an integral role in the activation of these MAPK proteins (11.Wilsbacher J.L. Juang Y.C. Khokhlatchev A.V. Gallagher E. Binns D. Goldsmith E.J. Cobb M.H. Biochemistry. 2006; 45: 13175-13182Crossref PubMed Scopus (26) Google Scholar). Studies with ERK2 have shown that disruption of ERK homodimers reduces its ability to localize to the nucleus and that only homodimers are actively transported to the nucleus (12.Adachi M. Fukuda M. Nishida E. EMBO J. 1999; 18: 5347-5358Crossref PubMed Scopus (242) Google Scholar, 13.Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). In addition, phosphorylation by upstream MAPK kinases is necessary for and precedes ERK2 dimerization (12.Adachi M. Fukuda M. Nishida E. EMBO J. 1999; 18: 5347-5358Crossref PubMed Scopus (242) Google Scholar). Unlike ERK2, the JNK2α2 isoform is capable of phosphorylating itself and downstream substrates, such as c-Jun, without the presence of upstream kinases (14.Pimienta G. Ficarro S.B. Gutierrez G.J. Bhoumik A. Peters E.C. Ronai Z. Pascual J. Cell Cycle. 2007; 6: 1762-1771Crossref PubMed Scopus (13) Google Scholar). This ability to autophosphorylate can occur in either a cis- (intra-) or trans- (inter-) molecular mechanism. Domain analysis has revealed that JNK2α2 autophosphorylation activity and its ability to translocate to the nucleus is dependent on the alternatively spliced region known as the α-region (15.Cui J. Holgado-Madruga M. Su W. Tsuiki H. Wedegaertner P. Wong A.J. J. Biol. Chem. 2005; 280: 9913-9920Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This 9-amino acid domain (LVKGCVIFQ) is critical for activation, because swapping domain the region of the active isoform, the isoform active (15.Cui J. Holgado-Madruga M. Su W. Tsuiki H. Wedegaertner P. Wong A.J. J. Biol. Chem. 2005; 280: 9913-9920Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). of the JNK2α2 α-region with the region of JNK2α2 that the α-region is critical for activation, is region in JNK2α2 dimerization and autoactivation. Because JNK2α2 activity enhances tumor it is important to its of autophosphorylation and autoactivation. findings demonstrate that JNK2α2 has a of autophosphorylation and autoactivation. We that dimerization is dependent on the α-region and occurs independently of disruption of JNK2α2 dimerization through specific in the α-region in of of autophosphorylation, and These results that JNK2α2 is from other MAPK proteins because its is dependent on and that dimerization most likely precedes autophosphorylation. of the through JNK2α2 is constitutively active be of for and isoform specific that be used to with glioblastoma multiforme. of JNK2α2 encoding wild type JNK2α2 was of were by mutagenesis to the mutagenesis The are The JNK2α2 mutant were then of or The were as previously (15.Cui J. Holgado-Madruga M. Su W. Tsuiki H. Wedegaertner P. Wong A.J. J. Biol. Chem. 2005; 280: 9913-9920Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Cell and cell line and were in Dulbecco's modified Eagle's with and The to were to the and and were expressed in the and induced with for at The proteins were to the was to and in at of protein was to a in were in the and the protein were by the were and with was the molecular to the and from were as previously Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). of protein was by The were used for JNK that the Cell c-Jun Cell and for were in of to The protein were and as previously Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). The protein were of J. was on as previously M. N. E. 2000; PubMed Scopus Google Scholar). The were a scanning that protein were and in a In of the protein was in of the kinase and at for The were by protein and for (8.Tsuiki H. Tnani M. Okamoto I. Kenyon L.C. Emlet D.R. Holgado-Madruga M. Lanham I.S. Joynes C.J. Vo K.T. Wong A.J. Cancer Res. 2003; 63: 250-255PubMed Google Scholar). kinase were in a and of was were previously Cell were with of protein in of was to protein and then at for The were by protein and the for A. S. EMBO J. 2006; PubMed Scopus Google Scholar). The were then by by The of and was by from the the 2007; PubMed Scopus (26) Google Scholar, 2005; PubMed Scopus Google Scholar). were as previously that the cell were used of Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). of the the were by in of for Cell were in and in with and The of were by of The were in and the results were expressed as the were in in of and and a of in and The presence of was 10 The were in and the results were expressed as the JNK2α2 and previously have autophosphorylation and activation of the JNK2α2 isoform, is from the JNK1 and JNK3 genes (8.Tsuiki H. Tnani M. Okamoto I. Kenyon L.C. Emlet D.R. Holgado-Madruga M. Lanham I.S. Joynes C.J. Vo K.T. Wong A.J. Cancer Res. 2003; 63: 250-255PubMed Google Scholar). the by JNK2α2 be of because it in the of for the of of JNK activity are the phosphorylation of the and the ability to downstream such as expressed JNK2α2 possesses these two of we an in vitro kinase with expressed with We used an JNK that only JNK it is at and This demonstrated that autophosphorylate in the of an upstream on the We that the only a at by JNK2α2 mutants that phosphorylation at individual and or at This that only the wild type JNK2 be by only the JNK2α2 protein can the Because our used expressed we to its activity to JNK2α2 from cells. JNK2α2 we an in vitro kinase with and either JNK2α2, was in or the expressed We used an specific for c-Jun to the JNK2α2 was a of JNK2α2 in the in 10 of analysis revealed that the expressed a of c-Jun phosphorylation with the cellular JNK2α2, suggesting that expressed JNK2α2 has a specific activity cellular JNK2α2 JNK2α2 on the but MAPK family such as dimerize to activation, and phosphorylation occurs to dimerization (11.Wilsbacher J.L. Juang Y.C. Khokhlatchev A.V. Gallagher E. Binns D. Goldsmith E.J. Cobb M.H. Biochemistry. 2006; 45: 13175-13182Crossref PubMed Scopus (26) Google Scholar, M. Fukuda M. Nishida E. EMBO J. 1999; 18: 5347-5358Crossref PubMed Scopus (242) Google Scholar). We a series of mutants to the in JNK2α2 phosphorylation is necessary for dimerization, we a kinase JNK2α2 in the that is critical for in the was to an In vitro kinase protein that autophosphorylate the of the we used a JNK2α2 in the JNK2α2 α-region was for the α-region from the isoform, was previously shown to possess autophosphorylation activity (15.Cui J. Holgado-Madruga M. Su W. Tsuiki H. Wedegaertner P. Wong A.J. J. Biol. Chem. 2005; 280: 9913-9920Abstract Full Text Full Text PDF PubMed Scopus (31) Google autophosphorylation activity in these JNK2α2 in JNK2α2 dimerization in its we used Previous has shown that JNK2α2 as a and a (11.Wilsbacher J.L. Juang Y.C. Khokhlatchev A.V. Gallagher E. Binns D. Goldsmith E.J. Cobb M.H. Biochemistry. 2006; 45: 13175-13182Crossref PubMed Scopus (26) Google Scholar). of wild type JNK2α2 in two distinct at and or a was protein We that the proteins of in with a JNK2α2 whereas the proteins of in with a JNK2α2 and Interestingly, the kinase mutant revealed two distinct at and and the was to the wild type JNK2α2 of the mutant also a as JNK2α2 These findings that JNK2α2 dimerization is dependent autophosphorylation. of only protein at indicating that the α-region is necessary for JNK2α2 to the of the we also a We used a primary that has been shown to the of protein A. S. EMBO J. 2006; PubMed Scopus Google Scholar). wild type JNK2α2 protein was with of the We found that the of was to a JNK2α2 we our studies to we found that a of was necessary to the JNK2α2 The is most likely an because a of was to the In addition, the presence of the is likely an because of in two The kinase mutant to wild type JNK2α2, that dimerization is our findings suggest that the but phosphorylation, is necessary for JNK2α2 JNK2α2 in the α-region is necessary for JNK2α2 dimerization in we and JNK2α2 in and We that the two JNK2α2 proteins suggesting that JNK2α2 dimerize in vivo we our studies to and mutant the α-region from we found that only the and be with and These results suggest that the α-region from JNK2α2 is necessary for dimerization in The were then in a glioblastoma cell we found that the wild type JNK2α2 interact with the whereas interact with revealed that more that it is the of the α-region from JNK2α2 that enhances the of JNK2α2 to In addition, the mutant with it that with the WT, suggesting that phosphorylation may to JNK2α2 dimerization in decrease was likely to in the because a revealed of mutant to the protein show that dimerization occur independently of phosphorylation, we also the JNK2α2 is incapable of at the and We found that the JNK2α2 mutant was at to the suggesting that phosphorylation JNK2α2 dimerization these findings suggest that JNK2α2 may with other JNK isoforms in it is the and phosphorylation, homodimers cells. of JNK2α2 our of the role that the α-region in JNK2α2 we an mutagenesis of the α-region of the amino were to an to amino are necessary for JNK2α2 autophosphorylation We only because and are In vitro kinase demonstrated that five mutants (L218A, K220A, G221A, I224A, and F225A) whereas only mutants and autophosphorylation activity the of autophosphorylation activity with an to we mutant and the We that autophosphorylation mutant (L218A, K220A, G221A, I224A, and F225A) as a and more to the and the mutants that the ability to autophosphorylate and as and and only the of to the JNK2α2 and These findings with the mutant suggest that JNK2α2 autophosphorylation is dependent on dimerization and that the amino and play an important role in for JNK2α2 to the dimerization is necessary for JNK2α2 autophosphorylation, it may also be important for other of JNK2α2 of JNK2α2 activity is the ability to translocate to the nucleus. the autophosphorylation and mutants were also in the ability to localize to the we mutants cells. a in of the with (15.Cui J. Holgado-Madruga M. Su W. Tsuiki H. Wedegaertner P. Wong A.J. J. Biol. Chem. 2005; 280: 9913-9920Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). of the mutants revealed that mutant (L218A, K220A, G221A, I224A, and F225A) only of the with a whereas the mutants and with a and In addition, analysis of the kinase mutant revealed that only of the a that are preferentially to the nucleus analysis an specific for JNK revealed that autophosphorylation mutant a in phosphorylation, whereas the mutants only a These results that is dependent on JNK2α2 for of JNK2α2 in has been previously shown to cellular proliferation and cell growth, whereas overexpression of the inactive JNK2α2 mutant these (9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google Scholar). We dimerization a role in the tumorigenic we used a to that expressed WT, and We the cellular growth of the cell in with This of was previously shown to the cellular growth of the growth that activate JNK (9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google Scholar). with the a growth with the (9.Cui J. Han S.Y. Wang C. Su W. Harshyne L. Holgado-Madruga M. Wong A.J. Cancer Res. 2006; 66: 10024-10031Crossref PubMed Scopus (65) Google The cell and to the and also a growth the other the were found to at a and a growth as cells. of growth of the cell with the the and cell all more whereas the a of to the cell line Together, these findings demonstrate that JNK2α2 dimerization is necessary for its cellular activity as as its ability to for JNK2α2 the of JNK dimerization, we the from our analysis of JNK2α2 JNK2α2 dimerization is the of a two the is a then be of the two in the protein to a JNK2α2 and This that JNK2α2 are and occur through a such as a This is by the that the JNK2α2 dimerization or activity studies suggest that JNK dimerization may be dependent on a leucine leucine zipper is of two that have by amino The from to a on a ERK2 and c-Jun were found to through a leucine zipper (11.Wilsbacher J.L. Juang Y.C. Khokhlatchev A.V. Gallagher E. Binns D. Goldsmith E.J. Cobb M.H. Biochemistry. 2006; 45: 13175-13182Crossref PubMed Scopus (26) Google Scholar, J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the of revealed that an may the indicating that the the α-region may be a component of a leucine zipper W. Su 1998; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, D. Wang S. S. D. M. J. A. J. Mol. Biol. PubMed Scopus Google Scholar). is our that the and prevent dimerization, autophosphorylation, and JNK2α2 activity additional for the of a leucine zipper in JNK2α2 dimerization, we isoleucines and leucines that the α-region and the (I208A, I214A, and a we also because it the and the leucine zipper D. Wang S. S. D. M. J. A. J. Mol. Biol. PubMed Scopus Google Scholar). In vitro kinase revealed that mutant the autophosphorylate or and the was found to the ability of JNK2α2 to autophosphorylate or our results suggest that JNK2α2 dimerization occurs in a and that it may be to a leucine a a of the JNK2α2 JNK2α2 in a are two for JNK2α2 undergoes is a that a cis- autophosphorylation or the kinases of the JNK2α2 other in trans- autophosphorylation we wild type JNK2α2 with the kinase-inactive the two proteins, a was to the in a whereas a was to the wild type JNK2α2 protein of the was to of wild type JNK2α2 autophosphorylation occurs in then of the phosphorylation of wild type is a phosphorylation then the kinase mutant prevent wild type phosphorylation In vitro kinase that a of with wild type JNK2α2 a decrease in wild type phosphorylation, and a in an decrease This that in a with a that JNK2α2 autophosphorylation occurs in we also at the ability of wild type JNK2α2 to the kinase JNK2α2 autophosphorylation occurs in then only the wild type JNK2α2 be but is a phosphorylation, then the mutant and wild type protein be We that the kinase mutant and wild type protein were the proteins were at a a a of with wild type JNK2α2 in an decrease in phosphorylation for these results is that the kinase mutant is incapable of with wild type JNK2α2 at we for the different of the We that of was to more wild type JNK2α2, suggesting that more are at Because of more phosphorylation may be our findings a dimerization precedes also a of JNK2α2 phosphorylation In we have the mechanisms to the activity of and we demonstrated that a 9-amino acid region known as the is necessary for JNK2α2 amino in the α-region are important for dimerization, we an mutagenesis different mutants were and through the of and we that five mutants (L218A, K220A, G221A, I224A, and F225A) of these mutants also its autophosphorylation activity as as its ability to localize to the nucleus. These findings suggest that JNK2α2 activity is dependent on a cell line cell proliferation or growth, indicating that dimerization is also necessary for JNK2α2 induced of the of JNK2α2 dimerization revealed dimerization occurs independently of JNK2α2 in a suggesting that the are constitutively and in vitro kinase with wild type JNK2α2 and a kinase mutant demonstrate that wild type JNK2α2 can the that JNK2α2 autophosphorylation occurs in a a mutagenesis of isoleucines and leucines the α-region (I208A, I214A, and in of JNK2α2 dimerization, indicating that may occur through a leucine the α-region also dimerization, and we that these the of the we have our of the mechanisms of JNK2α2 and we have shown that dimerization is important for tumorigenic phenotypes. of JNK2α2 findings suggest that JNK2α2 undergoes a wild type JNK2α2 the mutant and as a We that of more with wild type This a for the in JNK2α2 phosphorylation of also the phosphorylation, suggesting a for JNK2α2 we that the the JNK2α2 The other JNK2α2 to increased of JNK2α2 of more and the decrease in wild type and phosphorylation This most likely be because our analysis only JNK2α2 and The of MAPK of our MAPK dimerization is on ERK2 studies in and human cell have shown that ERK2 as a and a but phosphorylation ERK2 from the MAPK kinase and homodimers (12.Adachi M. Fukuda M. Nishida E. EMBO J. 1999; 18: 5347-5358Crossref PubMed Scopus (242) Google Scholar, 13.Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). in vitro studies have demonstrated that ERK2 kinase activity is dependent on dimerization because its kinase activity is and mutants have kinase activity as the wild type protein E. M. S. D. C. A. Cobb M.H. J. PubMed Google Scholar). in vivo have the of dimerization, because only ERK2 homodimers are actively transported to the and disruption of ERK2 dimerization by mutagenesis reduces its A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). MAPK proteins are to regulate MAPK family p38, was also shown to but it is its is A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). and JNK an but by or and JNK translocate to the that MAPK proteins are also actively transported J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Nishida E. 2005; PubMed Scopus Google Scholar). Studies the that the of JNK, is actively transported to the nucleus by a of the F. P. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). we show that JNK2α2 dimerization is necessary for activation in vitro and in vivo and that translocation is dependent on homodimerization. studies are to protein such as are for JNK2α2 to the nucleus. JNK2α2 the of have to for as of the JNK are such as This was found to by and from the of JNK as M. N. H. R. N. J. 2004; PubMed Scopus Google Scholar). has been with the have because of was shown to inhibit of protein suggesting that occur because of of other kinases J. H. M. P. J. 2003; PubMed Scopus Google Scholar). specific kinase to JNK be to of these work that JNK2α2 dimerization only inhibit JNK2α2 autophosphorylation and but it may also the of a more JNK because MAPK family has necessary for dimerization (11.Wilsbacher J.L. Juang Y.C. Khokhlatchev A.V. Gallagher E. Binns D. Goldsmith E.J. Cobb M.H. Biochemistry. 2006; 45: 13175-13182Crossref PubMed Scopus (26) Google Scholar, J. PubMed Scopus Google Scholar). of protein kinases have been to in a protein that a inhibit JNK2α2 homodimerization. We demonstrated the of JNK2α2 dimerization by the ability of our active and inactive mutants to active mutant and to wild type JNK2α2 and two tumorigenic cellular proliferation and growth and Interestingly, the inactive mutant was found to exist as a and is constitutively these phenotypes. These results suggest that or dimerization may decrease the tumorigenic ability of the role of JNK2α2 in glioblastoma a specific that JNK2α2 dimerization be important in the of tumor with