Isolation of Hyperactive Mutants of the MAPK p38/Hog1 That Are Independent of MAPK Kinase Activation

Abstract

Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38α, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions. Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38α, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions. mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun NH2-terminal kinase MAPK kinase hemagglutinin glutathione S-transferase MAPK1 is a generic term for a large family of enzymes, which function in a variety of signal transduction pathways. Mammalian MAPKs are divided into at least three subfamilies (ERKs, p38s, and JNKs) based on degree of homology, biological activities, and phosphorylation motif (1Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, 2Cobb M.H. Goldsmith E.J. Trends Biochem. Sci. 2000; 25: 7-9Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 3Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1381) Google Scholar, 4Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (899) Google Scholar, 5Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 6Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2274) Google Scholar). Although highly homologous in structure and in pattern of activation, each MAPK is activated in response to a specific battery of signals and in turn phosphorylates a particular array of substrates. As a result, each MAPK imposes specific effects on the cell. For example, in many cell lines (e.g. fibroblasts), the ERK MAPKs are activated when cells are exposed to growth factors, and their activation is important for enhancement of cell proliferation (7Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 8Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar). In other cells (neuronal and myogenic cell lines), however, activation of ERKs is associated with growth arrest and differentiation (7Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 9Gredinger E. Gerber A.N. Tamir Y. Tapscott S.J. Bengal E. J. Biol. Chem. 1998; 273: 10436-10444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In contrast to the ERK enzymes, the activity of p38 and JNK MAPKs is only slightly induced by growth factors. These enzymes are strongly activated in response to stress signals such as heat shock, osmotic shock, UV radiation, cytokines, and metabolic inhibitors. JNK and p38 MAPKs seem to be responsible mainly for protective responses, stress-dependent apoptosis, and inflammation (3Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1381) Google Scholar, 10Ono K. Han J. Cell. Signalling. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar). In some cell types, however, p38 and JNK may play a role in differentiation and development (3Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1381) Google Scholar, 10Ono K. Han J. Cell. Signalling. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar, 11Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-F104PubMed Google Scholar). Studies with knockout mice and knockout cell lines revealed essential roles for MAPKs in various aspects of embryonal development (12Pages G. Guerin S. Grall D. Bonino F. Smith A. Anjuere F. Auberger P. Pouyssegur J. Science. 1999; 286: 1374-1377Crossref PubMed Scopus (543) Google Scholar, 13Sabapathy K. Hu Y. Kallunki T. Schreiber M. David J.P. Jochum W. Wagner E.F. Karin M. Curr. Biol. 1999; 9: 116-125Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Yang D.D. Conze D. Whitmarsh A.J. Barrett T. Davis R.J. Rincon M. Flavell R.A. Immunity. 1998; 9: 575-585Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 15Dong C. Yang D.D. Wysk M. Whitmarsh A.J. Davis R.J. Flavell R.A. Science. 1998; 282: 2092-2095Crossref PubMed Scopus (534) Google Scholar, 16Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1542) Google Scholar, 17Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Although many aspects of MAPK biology have been revealed, the exact role of each MAPK in a given biological system is not fully understood and is difficult to study. The main reason for this difficulty is our inability to activate a given MAPK in vivo and to follow the biochemical and physiological consequences. Currently, a MAPK is experimentally activated in vivo using extracellular stimuli or through expression of an active form of a component that functions upstream to that MAPK (7Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 8Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar). Each of these treatments activates more than one MAPK and evokes many cellular responses. Activation of a given MAPK per se could be obtained theoretically by expressing an active form of this kinase, which would be active independently of external signals and upstream components. However, the catalytic activity of MAPKs is tightly regulated and strictly dependent on upstream activation (2Cobb M.H. Goldsmith E.J. Trends Biochem. Sci. 2000; 25: 7-9Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 18Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. S. M. Han J. R.J. Davis R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although the of MAPK activation have been revealed this could not be applied for the production of active forms of Here we describe a novel genetic which and such active of cells to an extracellular the activity of the MAPK D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This activation is through a signal transduction that in phosphorylation of the MAPK by a MAPK kinase are dual kinases that MAPKs on particular Thr and Tyr This dual phosphorylation is the for the in MAPK MAPKs in the Thr or Tyr be activated D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Alexander M.C. H. J. 1994; PubMed Scopus Google Scholar). The of MAPK activation dual the in active forms of these enzymes, a structure is difficult or to by D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the structure of with that of A. Cobb M.H. Goldsmith E.J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google that phosphorylation of the activation in the activation and in domain at the known as These the phosphorylation and the L16 that these an for of the kinase The is by mainly at L16 of the and by an and in of one and of the other J. M. M. Goldsmith E. Cobb M.H. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It is not known other MAPKs (e.g. and are phosphorylation and Although the of and revealed the that activate the not a for a active MAPK by have a novel for isolation of active forms of MAPKs using a genetic screen in The which are highly homologous to their (1Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, Trends Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The MAPKs and are to the ERK The Hog1 MAPK a phosphorylation motif to that of and is by Davis R.J. Science. 1994; 265: PubMed Scopus Google or J. R.J. Science. 1994; 265: PubMed Scopus Google Scholar). Hog1 is and activated by the MAPKK J. T. E. M. Science. PubMed Scopus Google a of A. A. H. C. F. Johnson G.L. Karin M. Science. PubMed Scopus Google Scholar). The MAPK is essential for of cells osmotic cells or on with of or J. T. E. M. Science. PubMed Scopus Google Scholar). to active forms of MAPKs of the that be to screen a of in cells The is that a that cells to on an MAPKK-independent Hog1 to active forms of MAPKs were only mutations that were in the S. Mol. Biol. Cell. 1994; PubMed Scopus Google Scholar, J.P. E. M.C. E. Mol. Cell. Biol. PubMed Scopus Google and in the MAPK of D. J. E. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, Marshall C.J. 1994; PubMed Scopus Google not the kinases The of M.J. Goldsmith E. Cobb M.H. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google C. J. T. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google more Yet, in the in vivo and MAPKs are not in the cell and are the of the be for physiological through various active forms of which are independent of MAPKK are not available. The in this is for MAPKs that are active in the of their In the of the screen just in the MAPK describe the isolation of nine point mutations in the Hog1 Each is to Hog1 and independent of upstream regulation. of mutations to the human rendered this enzyme active The S. used in this were the in which the The were obtained from M. The produced in this study. were on or on the and and and The of cells to osmotic on with are for each osmotic in were to of at were in by and in the or in were The of produced in to T. M. W. Scholar). the obtained from M. introduced into were obtained and to on for were in a into of and for the and for to by or with the to the of the The of the used for are in of used in and in a of the of mutants into as by and Curr. Genet. PubMed Scopus Google Scholar). cells were on that per were of and for by of on the to for and single These single were as as were in of to an of and by of the cells were induced for with the other of the cells were in cells were and in and of the were D. E. Karin M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, A. D. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). cell were to of of the induced with for as were and in of and of the were were in of at and of were Each for each were to and were with of of were in of by of of were for and for to The and for of the protein and the Hog1 were as in J. E.F. T. Scholar). The for the is of For of the were used The and were cell were to of a with as cells were at and the with of the in of of were and the for each were at for and the at for at were to and in For of the Hog1 of protein were with and with of at with and one with the were in and of the were were for at three with by three with kinase were The kinase by the in of kinase of and of as a The on the for the Hog1 in kinase and to are The at for and by the to and of to were through The were and exposed to and active mutants of were in Escherichia using the expression that a in their of and protein were to R.J. Han J. Cobb M.H. Goldsmith E.J. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). were as Y. A. K. R.J. Han J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google using as a screen for active forms of MAPKs of which the MAPKK and osmotic of the MAPK Hog1 not cells to on in each the of Hog1 activity on phosphorylation. This forms the for our that only an independent form of Hog1 to on The is to a that one or a mutants would intrinsic catalytic which is independent of The active mutants be by the to cells and on Each that an Hog1 kinase of mutants produced T. M. W. Scholar, T. J. R. K. M. J. PubMed Scopus Google and introduced into a J. T. E. M. Science. PubMed Scopus Google Scholar). were obtained and to on with with were to with that on of or were The growth on and the through a and by from to these not and were that the Hog1 mutants not to on of of of the and were not to cells when from a and an these mutants were only in some of the and were not to cells when from a and an these mutants were only in some of the For and were not to cells when from a and an these mutants were only in some of the in a The were Each to a single point in the of Hog1. of the an that the screen nine point mutations were and that each of these point mutations is to Hog1 independent of we through each to a and the mutants in nine mutations growth on showing that each point is to Hog1 independent of The that the variants in the screen are independent of MAPKs may be active as (2Cobb M.H. Goldsmith E.J. Trends Biochem. Sci. 2000; 25: 7-9Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, J. M. M. Goldsmith E. Cobb M.H. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google the that the mutants are not independent with the Hog1 that is in the the of the active mutants to knockout to on this is not by of Hog1 or Yet, is by the Hog1 mutants showing that are active independently of and Hog1. that the active Hog1 mutants cells by activating the biochemical to we of and The of these are in and their expression is (1Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar). revealed that the Hog1 active not induced of these in cells not the of the mutations the of the nine mutations are in a of and which is of the L16 These to be in to not the domain by and T. M. T. E. Cell Biol. 2000; PubMed Scopus Google Scholar). The other mutations are at the is just from the The in the is of the with the of MAPKs that mutations in that are conserved in at least one Hog1 variants that function independently of we to the biochemical of their The would be that these molecules have intrinsic catalytic directly kinase each and in for to mutants catalytic activity in the of stimulation these the activity of Hog1 molecules kinase activity for and The activity of these molecules not when cells were with and their catalytic activity growth and could be as active of Hog1 were used in is that the specific activity of the mutants is than that of Hog1. only and the other mutants catalytic activity in the of Yet, the activity of the other mutants increased when to the and mutants are hyperactive growth their activity that the in activity of some mutants a of of Hog1 a of a In Hog1 mutants in the screen are hyperactive independently of specific activity is way the activity of the activity of some mutants could be even induced by an that the mutants have independent catalytic we to as in E. to their Yet, we were not to Hog1 of Hog1 are difficult to were not in we to the activating mutations in the human which is known to be when in This is by the that the activating mutations of in that are conserved in MAPKs we of the human of in with or The and as as a p38 were in E. the and using The were for their kinase As p38 catalytic The however, kinase activity least than the p38 enzymes are active as in is that have intrinsic catalytic which is independent of activation by upstream the with the p38 mutants the that mutations in the screen could be used to active forms of the of the in kinase we a of This only when active p38 mutants were used this protein is an we the activity of the that and have the intrinsic for The activity of the mutants is mainly as by not phosphorylation on on and p38. an in is the responsible for the of the active variants of upstream activity of p38 The that the Hog1 active mutants an increased intrinsic catalytic This is strongly by the with the active p38 which were not activated by E. Although that the mutants have intrinsic we to the that the Hog1 mutants are vivo an to which phosphorylates and activates at least MAPK function in (1Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, Trends Genet. 1998; Full Text Full Text PDF PubMed Scopus Google be at least in the this we by the phosphorylation of the Hog1 used that Hog1 H. G. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar). cell were from and cells expressing Hog1 or the various were from cells or not to The revealed phosphorylation when the active Hog1 mutants and phosphorylation Hog1 mutants and of phosphorylation This of phosphorylation is even the in Hog1 in exposed to osmotic for the of Hog1 phosphorylation in cells could be activity Mol. Biol. Cell. 1994; PubMed Scopus Google Scholar, Y. A. K. R.J. Han J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It be that this phosphorylation of Hog1 in cells to growth on these that the dual phosphorylation of the active mutants is not to their intrinsic catalytic This been addressed by the and in the mutants Hog1 mutants a pattern of phosphorylation when in cells mutants and are in the of at a than that of the Hog1. the of their phosphorylation with is not than that of the of important for increased phosphorylation of these mutants and not for their biological activity mutants and in to be As in and and and in from cells expressing or a protein with the the of Hog1 is The only when some were used and not for for and and in vivo are to when are It is that these mutations induced which are responsible for the catalytic activity of these that and molecules are fully in cells and growth on In the that in cells mutants are not activated through dual phosphorylation, the and that strongly that the mutants an intrinsic independent catalytic The strongly that the active Hog1 mutants not phosphorylation for their Yet, some that phosphorylation, which may not be in the could activation Y. P. G. U. A. Marshall C.J. S. J. 1994; PubMed Scopus Google we to this this we in each of the active variants the and that mutants in particular and which are not at would of with and with and would As be in of with on the of mutants to growth of cells cells for and were by the mutation. This that phosphorylation of is for the activity of the mutants and the that are independent of of phosphorylation by in Hog1 and mutants their activity in cells and in cells of only with not the activity of with one The are that were when we to the mutants their to cells when and were to and of the mutants could growth on These that Hog1 mutants not dual phosphorylation for their activity on It is that phosphorylation of is not this an essential role in the catalytic of the not only D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google the in with not an in in a in the kinase is that the Hog1 mutants activity that phosphorylates the active Hog1 mutants were as molecules that cells to on are by In to their to growth osmotic we that expression of the active forms in cells important biological of the The growth growth of cells expressing the mutants The of these than that of cells expressing Hog1. In the active mutants increased cell and not of the induced by the mutants be in These effects novel roles for Hog1 in growth arrest and and for the of the active molecules for biological Although MAPKs are in pivotal biological processes and are been difficult to the exact role of a given MAPK in a particular biological a question could be using active forms of MAPKs that were not available. This a novel genetic screen in that active MAPK molecules that function independently of their The this screen is that only an active form of a MAPK would the in a MAPKK This applied for isolation of active Hog1 The mutants could Hog1 biochemical and biological independently of the active Hog1 variants the growth and other of the novel biological of Hog1. to these functions from the intrinsic catalytic activity The of the mutants for the of MAPKs in is by the that of mutations into the human kinase rendered with cells in that the active p38 mutants are highly active in to the Hog1 mutants in the with open the way to more active forms of various MAPKs based on the mutations in Hog1. of mutations into JNK to mutations and and into to the mutations and may these MAPKs that these mutations in a variety of MAPKs could a battery of active forms of each In to particular mutations that may MAPKs the mutations in our point the that be in an to activate Six of the nine mutations were in L16 and This domain is to in which to be important in the for J. M. M. Goldsmith E. Cobb M.H. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It is not known Hog1 or p38 are active as It could however, that the activating mutations mutations that a with a of and the of an active for this from the mutation. is homologous to in which which forms an with of J. M. M. Goldsmith E. Cobb M.H. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The that each a is that mutations on the MAPK would an even more and more active are mutations on the Hog1 and that in to the intrinsic activity of the mutants is that the mutations the L16 and the phosphorylation of the may in the phosphorylation from the to the open structure A. Cobb M.H. Goldsmith E.J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of the mutants (e.g. and are than and activity in the activity of other mutants increased by could be responsible for this in activity of some mutants when is It may be that activation of Hog1 requires the of an in to phosphorylation. In such a the in the activity of the mutants is a of this This may be by that of protein M. J. A. Davis R.J. Science. 1997; PubMed Scopus Google and M. M. C.J. Davis R.J. J. 1999; PubMed Scopus Google Scholar). It may be that the of of some active mutants their from an in with an in their catalytic that more than one the activity of the mutants may form may The in the intrinsic catalytic of the various mutants the of many are to fully the of of each In which are in the in protein induced by each mutation. may however, that on the Hog1 a that one with a that may an enzyme that is even more vivo than the molecules As our in this this the that applied for isolation of active Hog1 molecules could be used to screen for other active For example, active forms of could be in cells the MAPKK using the as the biological active forms of could a in one a screen for active forms of MAPKs in For example, p38 and JNK are in Davis R.J. Science. 1994; 265: PubMed Scopus Google Scholar, J. R.J. Science. 1994; 265: PubMed Scopus Google be to screen for active forms of these enzymes directly in However, the with that for active forms of other MAPKs may not be The activating mutations in Hog1 could be applied directly in many other M. for and Cobb for on p38 expression and for for and the and and for the