ERK3 Is a Constitutively Nuclear Protein Kinase

The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp) expected to be involved in catalysis eliminated autophosphorylation. Ser of ERK3, which corresponds to Thr, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions. The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp) expected to be involved in catalysis eliminated autophosphorylation. Ser of ERK3, which corresponds to Thr, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions. INTRODUCTIONERKs (extracellular signal-regulated protein kinases) 1The abbreviations used are: ERKextracellular signal-regulated protein kinasesMAPmitogen-activated proteinFBSfetal bovine serumDTTdithiothreitolPCRpolymerase chain reactionGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluorideHAhemagglutininPAGEpolyacrylamide gel electrophoresis.are a subfamily of the protein kinases involved in hormonal signal transduction. Two closely related members of this subfamily, the mitogen-activated protein (MAP) kinases ERK1 and ERK2, are regulated by growth factors and products of the protooncogenes Ras and Raf (reviewed in (1.Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 2.Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 19: 236-239Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 3.Cobb M.H. Goldsmith E. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar)). Blockade of the MAP kinases using dominant interfering mutations inhibits growth factor and oncogene-induced cell proliferation(4.Sontag E. Federov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (459) Google Scholar). Other ERK relatives include the c-Jun N-terminal protein kinase/stress-activated protein kinases, which phosphorylate and activate c-Jun(5.Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar, 6.Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar), and p38(7.Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2402) Google Scholar, 8.Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1494) Google Scholar, 9.Lee J.C. Laydon J.T. Mcdonnell P.C. Gallagher T.F. Kumary S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3121) Google Scholar), which is required for lipopolysaccharide-induced translation of tumor necrosis factor(7.Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2402) Google Scholar). These enzymes have been suggested to play a role in the response of cells to stress. ERK3, the third member of the ERK family to be cloned(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar), is 50% identical to ERKs 1 and 2 within its catalytic domain. Its cDNA from rat predicts a protein of 62 kDa with a C-terminal domain that extends 180 amino acids beyond the conserved ERK core. Given the important roles of other members of this subfamily of protein kinases in cellular regulation, it is likely that ERK3 will also have important, albeit unknown, functions.A human ERK3-related enzyme was cloned (11.Gonzalez F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar) that is 72% identical to rat ERK3 in the kinase domain. The tail domains of ERK3 and the ERK3-related enzyme are less similar (only 28% identical) and are unrelated over the last 65 amino acids. More recently, Flier and colleagues have found the human ERK3 homolog. It is nearly identical to rat ERK3 throughout the catalytic core as well as the tail, and appears to be alternatively spliced, generating a form that contains an additional 178 residues at the C terminus(12.Zhu A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar). Southern analysis suggests at least three ERK3-related genes, supporting the idea that there are multiple ERK3-like proteins(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar). One unusual feature of ERK3 and the related kinase is that they contain an arginine in place of the subdomain VIII glutamate that is highly conserved among the protein kinases(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar, 11.Gonzalez F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar, 12.Zhu A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar, 13.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3783) Google Scholar). Analysis of crystal structures of several protein kinases including ERK2 suggests that this residue is involved not in catalysis but in stabilizing the structure of the C-terminal fold of the protein kinase(14.Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1439) Google Scholar, 15.Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar). Members of the casein kinase I family also lack this glutamate(16.Rowles J. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9548-9552Crossref PubMed Scopus (121) Google Scholar). The three-dimensional structure of a yeast casein kinase I indicates a different mechanism to stabilize the protein structure(17.Xu R.-M. Carmel G. Sweet R.M. Kuret J. Cheng X. EMBO J. 1995; 14: 1015-1023Crossref PubMed Scopus (181) Google Scholar), suggesting that this arginine in ERK3 is compatible with its protein kinase activity. To test this directly, we expressed and immunopurified ERK3 to its catalytic we to the of ERK3 protein by its and in cells and tissues. antibodies that ERK3 was unlike ERK1 and ERK2, ERK3 was localized to the nucleus in the of and cells at in a in with bovine and 2 The cell was in with the cells in for and with growth factor or for and of ERK3 and ERK3 the tail domain the of ERK3 was by chain and and sites of which to the The was and to protein as D.J. E. C. D. Cobb M.H. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). Recombinant protein was by to and with a of in ERK T.G. J.S. Cobb M.H. 1991; PubMed Scopus Google Scholar). The and to of 1 1 1 and ERK and at ERK3 as a used for of or ERK3 and the products and sites of J.E. Biochem. 1991; PubMed Scopus Google Scholar). was as that in J. T. Scholar) to an of and with for with and and in of 1 1 1 1 1 2 and The for 2 to and by at for in a The with for 2 and the with 1 1 1 and with ERK and at of with peptides from the C-terminal tail or protein kinase subdomain of ERK3 peptides by of as T.G. Cobb M.H. 1991; PubMed Scopus Google Scholar). The from the kinase antibodies in and The from the C-terminal antibodies in and Two other peptides from the C and also used to antibodies in of and and was also used as E. D. Scholar) to antibodies in and was by the antibodies with or recombinant ERK3 as with the rat in of 1 2 1 and 2 by and with of a over of the and at in a for at by the in 1 of for and at in a for 1 and cells and as C. J. Mol. Cell. Biol. 1992; PubMed Google Scholar, R.M. PubMed Scopus Google Scholar). with 1 1 1 1 2 of protein from to gel by with of ERK3 was in to 1 and 1 at for with and by and T.G. Yancopoulos G.D. J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Science. PubMed Scopus Google Scholar). of by ERK3 was as D.J. E. C. D. Cobb M.H. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, T.G. Yancopoulos G.D. J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Science. PubMed Scopus Google and cells in by as C. J. Mol. Cell. Biol. 1992; PubMed Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) with or by or using a of ERK3 fragment was for with a in vitro was in its to that the been The ERK3 expressed in bacteria and mammalian cell lines by the as for ERK3 and of ERK3 in mammalian cell ERK3 or and sites of by of J.T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google to the or and sites of G. J.M. Mol. Cell. Biol. PubMed Scopus Google Scholar) to the using the was with or J. T. cells to bovine T.G. Yancopoulos G.D. J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Science. PubMed Scopus Google Scholar) for 1 to of for 2 with in ERK with 1 and for at for in an the cell with 2 and of protein for 2 at The three with 1 1 with and with 1 The by and was using the of R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). analysis and as D.J. Cobb M.H. Mol. Biol. Cell. 1992; PubMed Scopus Google of ERK3 by ERK3, we antibodies to one from subdomain of the catalytic three peptides within the kDa of the enzyme that C-terminal to the catalytic domain as well as to recombinant of the resulting a protein of the 62 in cell from and cells and not was by of the with the peptides not The from subdomain was identical to regions of ERK1 and of and of the from the C-terminal domain are also in the ERK3-related kinase, one of is from a in ERK3 ERK3 was from the related kinase, of in of the C-terminal domains that the catalytic core. The and a protein of the size as the other ERK3 indicating that the signal is from ERK3, not the related the and of ERK3 in rat using and the an to subdomain an of 62 to ERK3 that by antibodies also revealed a ERK3 with a distinct as well as of and to ERK1 and ERK2, with the of to ERK3 in subdomain and also of and kDa in rat suggesting the of ERK3-related in tissues. The protein be the alternatively form of ERK3 by A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar) it is found highly in of ERK3 in rat tissues. from rat and to for and used for The are of the of ERK3 by analysis of cells and Immunoblotting with the that ERK3 was in the of cells with in the with the the expected that ERK1 and ERK2 in the with in the T.G. Cobb M.H. 1991; PubMed Scopus Google Scholar). found for and cells of ERK2, and ERK3 in cells. and from cells or with to and with ERKs 1 and and ERK3 and to a ERK1 and of ERK3 was by with the ERK3 was found in the of cells and The was by the with C and or recombinant not signal in cells not also with several other cell including and cells. cells and cells to activate MAP kinase ERK3 was also in the nucleus and the of ERK1 and ERK2 was with ERK1 and ERK2 in the of they in and the nucleus C. J. Mol. Cell. Biol. 1992; PubMed Google Scholar) they also in the of ERK3 in cells. cells with the cells with J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). cells with with the cells with ERK3 in cells for as cells with for ERK1 and ERK2 with in cells for as cells with for for and C for and C-terminal tail of ERK3 has several that for its in the To the C-terminal domain was for the of ERK3, the protein and ERK3 the C-terminal 180 amino expressed in cells. The cells by with the and with cell the was found primarily in the nucleus of the cells was also in the nucleus as well as in the Immunoblotting of also that ERK3 and and in the of ERK3 is of its C-terminal and cells. in cells with for and the as for of cells as and and with and are The at 62 kDa in the is of expressed in ERK3 by and with the used to test the of ERK3 to phosphorylate in vitro of ERK1 and ERK3 phosphorylated of substrates, including and protein to a not It is that ERK3 phosphorylation to protein kinase activity. lack of phosphorylation is to for the of ERK3, ERK3 was at least in the phosphorylated form and its was from protein expressed in was also expressed in bacteria and cells to test a role of the C terminus of ERK3 its activity. form of ERK3 also with the of substrates, we the of ERK3 to autophosphorylate. ERK3 and that ERK3 is a protein conserved (Asp) important for catalytic S.K. Quinn A.M. 1991; PubMed Scopus Google Scholar) was to to not the that an kinase phosphorylated of of and in analysis of autophosphorylated and The are of the phosphorylation of ERK2 and ERK3 are The phosphorylation sites of ERK2 are with The identical residues ERK2 and ERK3 are marked with of and in analysis of autophosphorylated and The are and are of that ERK3 was an ERK3 autophosphorylated in a not ERK3 not phosphorylate this ERK3 was with ERK3 and ERK3 was with was not ERK3 and ERK3 not phosphorylated by ERK3 from cells with analysis that the ERK3 and and in the was less of of ERK3 of of ERK2 is D.J. E. C. D. Cobb M.H. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, D.J. Cobb M.H. Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar, T.G. Yancopoulos G.D. Panayotatos N. Radziejewska E. L. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, P. J. Hunt M.J. EMBO J. 1991; PubMed Scopus Google Scholar). and the residue are phosphorylated in the form of These sites in the phosphorylation and VIII of the protein kinases, a of ERK1 and ERK2, but different in The residue to is in ERK3, that to is Ser that ERK3 autophosphorylated we the that Ser was a Ser was to or To an ERK3 with the phosphorylation residues of ERK2, Ser and to and and ERK3 autophosphorylated at a to ERK3 ERK3, but the residue autophosphorylated was and one in autophosphorylated ERK3 or and was from autophosphorylated ERK3 but was by peptides from and ERK3 These that Ser is the of of ERK3 and of of autophosphorylated autophosphorylated autophosphorylated ERK3 and of autophosphorylated and ERK3 Ser in ERK3 is phosphorylated in and Ser is the phosphorylation and ERK3 expressed in with and with the ERK3 was phosphorylated and The phosphorylation of ERK3 was with suggesting that Ser is the phosphorylation of ERK3 in of ERK3 in cells. and cells. was from cell with and is analysis of phosphorylated The are and ERK3 cells. and the from cell with and is is a ubiquitously expressed in a variety of rat and mammalian cell Two human ERK3 have been one of which appears to be the of rat F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar, 12.Zhu A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar). The human is alternatively resulting in ERK3 of 62 and the of which has an additional 178 residues at its C a variety of cell the form was the form by with antibodies to different A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar) also found the form in cells by in rat one of also indicating that multiple ERK3-related are is as a member of the ERK subfamily of its subdomain of ERK3 is identical to ERK1 and subdomain is 72% subdomain of protein kinase is identical to is the in of conserved the other ERK3 appears to a distinct from the of the kinase of ERK3 it from other ERK ERK3 does not the activating phosphorylation a of this It does not phosphorylate by other MAP kinases. ERK3 is it is not in response to a it a to ERK1 and ERK2, in the phosphorylation other family does not phosphorylate MAP kinase or of other MAP kinase including as and the of ERK3 by as they have for other protein S. N. M.F. Biol. 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). ERK3 phosphorylated peptides in the that it has protein kinase activity. the a to be a of the phosphorylated and L. C. that ERK3 has a Mutation of of ERK2 to as in that of ERK3, the of ERK2 to phosphorylate its substrates, suggesting that the residue be a factor in protein in this kinase E. M. D. E. and M. in to the form of ERK3 in was to phosphorylate and A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar). One for this is that the form of ERK3 be a protein is that the ERK3 with protein have found at least one protein kinase that ERK3 and not ERK3 but also unlike ERK1 and ERK2, is localized to the nucleus in the of by and by The to that the protein was ERK3 its ERK3 appears to be to of it with and required or for It appears that the C-terminal 180 residues stabilize but are not required for its localization. ERK3 C-terminal residues a primarily of the protein is found in the be to a in its as the protein is 45 kDa and from the nucleus the a in autophosphorylated The of a in indicates that this is within the catalytic domain. Ser is the deduced phosphorylation the of the in an ERK3 this Ser in the phosphorylation and VIII and corresponds to the phosphorylation in the MAP kinases. suggests that ERK3 be regulated in a to other MAP kinase family ERK3 is phosphorylated by or but is phosphorylated by a in and of ERK3 this as well but phosphorylation of this in cells is likely to be by a enzyme as the one we have in and that ERK3 is not in the MAP kinase but in a distinct ERK3 have been it is not to test of Ser phosphorylation ERK3 activity. we have been to this phosphorylation its activity. are the of ERK3 that will to the and of this unusual protein INTRODUCTIONERKs (extracellular signal-regulated protein kinases) 1The abbreviations used are: ERKextracellular signal-regulated protein kinasesMAPmitogen-activated proteinFBSfetal bovine serumDTTdithiothreitolPCRpolymerase chain reactionGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluorideHAhemagglutininPAGEpolyacrylamide gel electrophoresis.are a subfamily of the protein kinases involved in hormonal signal transduction. Two closely related members of this subfamily, the mitogen-activated protein (MAP) kinases ERK1 and ERK2, are regulated by growth factors and products of the protooncogenes Ras and Raf (reviewed in (1.Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 2.Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 19: 236-239Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 3.Cobb M.H. Goldsmith E. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar)). Blockade of the MAP kinases using dominant interfering mutations inhibits growth factor and oncogene-induced cell proliferation(4.Sontag E. Federov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (459) Google Scholar). Other ERK relatives include the c-Jun N-terminal protein kinase/stress-activated protein kinases, which phosphorylate and activate c-Jun(5.Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar, 6.Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar), and p38(7.Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2402) Google Scholar, 8.Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1494) Google Scholar, 9.Lee J.C. Laydon J.T. Mcdonnell P.C. Gallagher T.F. Kumary S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3121) Google Scholar), which is required for lipopolysaccharide-induced translation of tumor necrosis factor(7.Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2402) Google Scholar). These enzymes have been suggested to play a role in the response of cells to stress. ERK3, the third member of the ERK family to be cloned(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar), is 50% identical to ERKs 1 and 2 within its catalytic domain. Its cDNA from rat predicts a protein of 62 kDa with a C-terminal domain that extends 180 amino acids beyond the conserved ERK core. Given the important roles of other members of this subfamily of protein kinases in cellular regulation, it is likely that ERK3 will also have important, albeit unknown, functions.A human ERK3-related enzyme was cloned (11.Gonzalez F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar) that is 72% identical to rat ERK3 in the kinase domain. The tail domains of ERK3 and the ERK3-related enzyme are less similar (only 28% identical) and are unrelated over the last 65 amino acids. More recently, Flier and colleagues have found the human ERK3 homolog. It is nearly identical to rat ERK3 throughout the catalytic core as well as the tail, and appears to be alternatively spliced, generating a form that contains an additional 178 residues at the C terminus(12.Zhu A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar). Southern analysis suggests at least three ERK3-related genes, supporting the idea that there are multiple ERK3-like proteins(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar). One unusual feature of ERK3 and the related kinase is that they contain an arginine in place of the subdomain VIII glutamate that is highly conserved among the protein kinases(10.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1478) Google Scholar, 11.Gonzalez F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar, 12.Zhu A.X. Zhao Y.I. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar, 13.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3783) Google Scholar). Analysis of crystal structures of several protein kinases including ERK2 suggests that this residue is involved not in catalysis but in stabilizing the structure of the C-terminal fold of the protein kinase(14.Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1439) Google Scholar, 15.Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar). Members of the casein kinase I family also lack this glutamate(16.Rowles J. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9548-9552Crossref PubMed Scopus (121) Google Scholar). The three-dimensional structure of a yeast casein kinase I indicates a different mechanism to stabilize the protein structure(17.Xu R.-M. Carmel G. Sweet R.M. Kuret J. Cheng X. EMBO J. 1995; 14: 1015-1023Crossref PubMed Scopus (181) Google Scholar), suggesting that this arginine in ERK3 is compatible with its protein kinase activity. To test this directly, we expressed and immunopurified ERK3 to its catalytic we to the of ERK3 protein by its and in cells and tissues. antibodies that ERK3 was unlike ERK1 and ERK2, ERK3 was localized to the nucleus in the of