The Direct Activation of MIK, a Germinal Center Kinase (GCK)-like Kinase, by MARK, a Maize Atypical Receptor Kinase, Suggests a New Mechanism for Signaling through Kinase-dead Receptors

Abstract

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs. Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs. Receptor protein kinases (RPKs) 1The abbreviations used are: RPKreceptor protein kinasesPRKplant receptor kinasesMAPmitogen-activated proteinMAPKMAP kinaseMAPKKMAP kinase kinaseDAPdays after pollinationRACErapid amplification of cDNA endsGSTglutathione S-transferaseHAhemagglutininMBPmyelin basic proteinIDintracellular domainGCKgerminal center kinase. are essential components of the cell regulation machinery that transmits extracellular signals to the inside of the cell. Although RPKs show high variability in their receptor domain, they share highly conserved cytoplasmic kinase domains, and they are assumed to function by a relatively well conserved general mechanism. After ligand binding and receptor oligomerization, the intracellular kinase domain becomes activated. This results in intermolecular auto-phosphorylation and conformational changes that allow the receptor to bind downstream signaling proteins (see Refs. 1Pawson T. Nash P. Genes Dev. 2000; 14: 1027-1047PubMed Google Scholar and 2Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2280) Google Scholar). Nevertheless, atypical RPKs that transduce signals by phosphorylation-independent mechanisms have been described recently (3Kroiher M. Miller M.A. Steele R.E. BioEssays. 2001; 23: 69-76Crossref PubMed Scopus (80) Google Scholar). They include phosphorylation-capable PRKs that can also signal through other mechanisms but also completely kinase-defective atypical RPKs, such as CCK-4 (4Mossie K. Jallal B. Alves F. Sures I. Plowman G.D. Ullrich A. Oncogene. 1995; 11: 2179-2184PubMed Google Scholar), H-Ryk (5Hovens C.M. Stacker S.A. Andres A.C. Harpur A.G. Ziemiecki A. Wilks A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11818-11822Crossref PubMed Scopus (116) Google Scholar, 6Katso R.M. Russell R.B. Ganesan T.S. Mol. Cell. Biol. 1999; 19: 6427-6440Crossref PubMed Scopus (62) Google Scholar), ErbB-3 (7Sierke S.L. Cheng K. Kim H.H. Koland J.G. Biochem. J. 1997; 322: 757-763Crossref PubMed Scopus (133) Google Scholar, 8Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway III, K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (596) Google Scholar), and DNT (9Savant-Bhonsale S. Friese M. McCoon P. Montell D.J. Gene (Amst.). 1999; 231: 155-161Crossref PubMed Scopus (13) Google Scholar). These atypical RPKs have substitutions within the kinase-conserved motifs, especially in the aspartic acid in subdomain VIb and in the DFG activation loop motif of subdomain VII. Among them, ErbB3 and H-Ryk are probably the best known examples. It has been shown that mice knockouts for ErbB-3 or Ryk present severe mutant phenotypes suggesting that both proteins are essential for signaling (10Riethmacher D. Sonnenberg-Riethmacher E. Brinkmann V. Yamaai T. Lewin G.R. Birchmeier C. Nature. 1997; 389: 725-730Crossref PubMed Scopus (605) Google Scholar, 11Halford M.M. Armes J. Buchert M. Meskenaite V. Grail D. Hibbs M.L. Wilks A.F. Farlie P.G. Newgreen D.F. Hovens C.M. Stacker S.A. Nat. Genet. 2000; 25: 414-418Crossref PubMed Scopus (145) Google Scholar). ErbB3 forms heterodimers with other members of the family of epidermal growth family receptors and is phosphorylated by these kinase-active RPKs (12Kim H.H. Vijapurkar U. Hellyer N.J. Bravo D. Koland J.G. Biochem. J. 1998; 334: 189-195Crossref PubMed Scopus (84) Google Scholar). Upon phosphorylation, ErbB3 serves as docking sites for multiple downstream signaling proteins. H-Ryk also forms heterodimers with other kinase-active RPKs, although in this case the interaction does not result in phosphorylation (13Trivier E. Ganesan T.S. J. Biol. Chem. 2002; 277: 23037-23043Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A chimeric receptor approach has shown that the ligand stimulation of H-Ryk results in activation of the MAPK pathway (6Katso R.M. Russell R.B. Ganesan T.S. Mol. Cell. Biol. 1999; 19: 6427-6440Crossref PubMed Scopus (62) Google Scholar), suggesting that activated H-Ryk can interact with and activate other downstream signaling proteins. Indeed, it has been proposed that signaling through atypical RPKs could involve regulated protein-protein interactions through their intracellular domains, which would explain the high conservation of the kinase-like structure of these domains through evolution (14Stein R.A. Staros J.V. J. Mol. Evol. 2000; 50: 397-412Crossref PubMed Scopus (79) Google Scholar). Nevertheless, the signal transduction mechanism for most atypical RPKs is still unknown. receptor protein kinases plant receptor kinases mitogen-activated protein MAP kinase MAP kinase kinase days after pollination rapid amplification of cDNA ends glutathione S-transferase hemagglutinin myelin basic protein intracellular domain germinal center kinase. MAP kinases play a key role on signal transduction in eukaryotes. MAP kinase modules are usually connected to the membrane-located receptors through G proteins, such as Ras or heterotrimeric complexes, or kinases that phosphorylate MAPKKK and are denoted as MAPKKKK (15Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5262Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 16Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 17Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Here we report the cloning and characterization of two maize genes coding for an atypical plant receptor kinase (PRK) and for a kinase of the GCK subfamily of MAPKKKK, which have been named MARK and MIK, respectively. We show that MARK and MIK interact in yeast, in vitro, and in mammalian cells. Moreover, phosphorylation experiments show that this interaction results in the induction of the kinase activity of MIK, suggesting a new mechanism for signaling through atypical receptor kinases. Plant Material—Zea mays cv. W64A plants were grown at 26 °C on a 16-h light/8-h dark cycle. Cloning of MARK and MIK cDNAs—A partial MARK cDNA was obtained from a differential screening of a 12-day after pollination (DAP) maize embryo cDNA library using 12-DAP embryo cDNA and leaf cDNA as probes, as described elsewhere (18Roca R. Stiefel V. Puigdomenech P. Gene (Amst.). 1998; 208: 67-72Crossref PubMed Scopus (12) Google Scholar). This clone was used to screen maize cDNA and genomic libraries (18Roca R. Stiefel V. Puigdomenech P. Gene (Amst.). 1998; 208: 67-72Crossref PubMed Scopus (12) Google Scholar) to obtain the full-length cDNA and genomic sequence. The full-length MIK sequence was obtained by RACE PCR on 15-DAP maize embryo cDNA with the primers MIKRACE1 (5′-CATCCATGCCTTTTGC-3′) and MIKRACE2 (5′-GATGCTTGAGAAGCTCCTTGG-3′) designed from the partial cDNA obtained from a yeast two-hybrid cDNA library (see below), using a RACE PCR kit (Invitrogen) according to manufacturer's instructions. Northern Analysis—Total RNA from immature seeds and adult leaves was obtained as described (18Roca R. Stiefel V. Puigdomenech P. Gene (Amst.). 1998; 208: 67-72Crossref PubMed Scopus (12) Google Scholar). 10 μg of the different RNAs was subjected to denaturing electrophoresis, blotted to nylon membranes (Nytran; Schleicher & Schuell), and hybridized with radiolabeled MARK or MIK cDNA probes following standard procedures as reported previously (18Roca R. Stiefel V. Puigdomenech P. Gene (Amst.). 1998; 208: 67-72Crossref PubMed Scopus (12) Google Scholar). Recombinant Proteins and Production of Antibodies—A partial cDNA corresponding to the last 325 amino acids of MARK protein, which coincide with its putative intracellular domain (ID-MARK), and the partial MIK cDNA corresponding to the last 143 amino acids of MIK (C-MIK) obtained from the yeast two-hybrid screening (see below) were cloned into a pET28 vector, and the recombinant His6-tagged ID-MARK and His6-tagged MIK were obtained as described (19Espinas M.L. Canudas S. Fanti L. Pimpinelli S. Casanova J. Azorin F. EMBO Rep. 2000; 1: 253-259Crossref PubMed Scopus (56) Google Scholar). ID-MARK cDNA and C-MIK cDNA were also cloned into a pGEX-KG expression vector (Amersham Biosciences) in order to obtain the GST-ID-MARK and GST-C-MIK as described (19Espinas M.L. Canudas S. Fanti L. Pimpinelli S. Casanova J. Azorin F. EMBO Rep. 2000; 1: 253-259Crossref PubMed Scopus (56) Google Scholar). Antibodies against ID-MARK and C-MIK were obtained in rabbits inoculated with the His6-tagged ID-MARK and His6-tagged C-MIK recombinant proteins. ID-MARK and MIK full-length cDNAs were cloned into pFLAG-CMV (Sigma) and pCMV5-HA vector (Clontech) in order to express both tagged proteins in mammalian COS-7 cells. Immunofluorescence Assays—For immunolocalization assays, 15-DAP immature maize embryos were fixed in an ethanol/formaldehyde/glacial acetic acid (80:3.5:5) fixative solution for 1 h at room temperature and for 7 days at 4 °C. Embryos were dehydrated and embedded in paraffin. 8-μm sections were deparaffinized with xylol, dehydrated, and permeabilized with 0.5% Triton X-100. Sections were incubated with the corresponding antibodies (1:200) in phosphate-buffered saline buffer with 2% bovine serum albumin, 0.1% Triton X-100, for 1 h at room temperature plus 1 h at 4 °C. After extensive washing sections were incubated with an anti-rabbit Cy3-conjugated antibody (Jackson ImmunoResearch) (1:400) in phosphate-buffered saline with 0.1% Triton X-100, 2% bovine serum albumin for 1 h. After extensive washing, sections were mounted with Mowiol and stored at 4 °C. Fluorescence was analyzed by confocal laser scanning microscopy (Leica TCS SP). Two-hybrid Analysis and GST Pull-down Assays—To construct the pAS2-1 bait plasmid, a partial cDNA corresponding to the last 325 amino acids of MARK was fused to the Gal4 DNA binding domain in plasmid pAS2-1 to generate pAS2-MARK (Clontech, Palo Alto, CA). This plasmid was used to screen a maize embryo (7 DAP) cDNA library cloned in the pACT2 plasmid kindly provided by Dr. Werr (Köln University). The screening was performed in strain Y190 of Saccharomyces cerevisiae (MATα, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, gal4Δ, gal80Δ, cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS, MEL1 URA::GAL1UAS-GAL1UAS-lacZ), and transformants were grown on selective medium lacking tryptophan, leucine, and histidine in the presence of to The activity of the was by using the the of yeast were with the clone obtained from the screening and pAS2-MARK or the full-length cDNAs of and cloned into the pAS2-1 plasmid that was used as The of the interactions was performed as described (19Espinas M.L. Canudas S. Fanti L. Pimpinelli S. Casanova J. Azorin F. EMBO Rep. 2000; 1: 253-259Crossref PubMed Scopus (56) Google Scholar). GST experiments were performed as described (19Espinas M.L. Canudas S. Fanti L. Pimpinelli S. Casanova J. Azorin F. EMBO Rep. 2000; 1: 253-259Crossref PubMed Scopus (56) Google Scholar) using the His6-tagged ID-MARK as an on different MIK GST proteins to were using the multiple 1994; PubMed Scopus Google Scholar). and in J. Scholar) were used to generate a on the and the corresponding respectively. were performed using the and from J. Scholar). maize embryos were in and proteins were with buffer and The was at and the was at for a (Amersham was in 10 and Triton buffer at were and of was Cell Cell and were in medium with serum and 10 were using (Invitrogen) according to the manufacturer's were h after in of buffer 0.5% 10 10 10 1 and 1 Cell were at for 10 of was with of a of protein (Sigma) and incubated with antibody (Sigma) or antibody (Sigma) for h at 4 °C. of a of protein (Sigma) was to the and incubated for 1 h at 4 °C. were in Cell or were by Proteins were with the was cloned into pGEX-KG expression vector and the was as MARK auto-phosphorylation and on myelin basic protein was performed as described K. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), using GST as and K. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) as a After phosphorylation, proteins were by The was with to and The was with a using the on proteins were performed in of phosphorylation buffer 1 10 μg of and 10 of (Amersham The protein kinase were performed at °C for and the were by of were subjected to with MARK, a with a of a to identify genes involved in maize we performed a differential screening of an embryo cDNA library with embryo and adult leaf cDNA of the putative cDNA high sequence with genes coding for kinases. This clone was for and used as a to screen embryo cDNA and genomic maize The cDNA and genomic an of amino acids with high sequence with PRKs We named this protein MARK maize atypical receptor As from the protein MARK a signal an extracellular domain with a domain, and an intracellular domain high sequence with kinases. Although the intracellular domain of MARK the conserved of some of the and highly conserved amino acids within these are the aspartic acid present in the subdomain which is assumed to of the kinase-active J. J.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), by an and the aspartic acid and within the DFG activation loop are by a acid and an and (see of the aspartic acid to relatively is to the kinase I. T. Oncogene. Google Scholar), and the role of the of the DFG has been in the case of H-Ryk (6Katso R.M. Russell R.B. Ganesan T.S. Mol. Cell. Biol. 1999; 19: 6427-6440Crossref PubMed Scopus (62) Google Scholar). This that the intracellular domain of MARK could an atypical with a We ID-MARK in as a with and we the of the recombinant protein to or in vitro. The results in show that ID-MARK not phosphorylate was it in the in which the intracellular domain of an with a intracellular kinase domain K. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), used as a and Cloning MIK, a PRKs are to in signal transduction by of regulated protein-protein interactions (3Kroiher M. Miller M.A. Steele R.E. BioEssays. 2001; 23: 69-76Crossref PubMed Scopus (80) Google Scholar). We for proteins that could interact with the intracellular domain of MARK using a yeast two-hybrid The screening of of a yeast two-hybrid immature embryo cDNA library using ID-MARK as bait a of of them coding for a with high sequence with the domain of two highly from S. A. M. 1999; PubMed Scopus Google Scholar). We named the corresponding MIK, from MARK kinase. The of interaction by a yeast two-hybrid is shown in growth was obtained the with the MIK partial corresponding to the most 143 amino acids of the ID-MARK in the corresponding pACT2 and was on selective medium at the and growth was the was at a the with the MIK partial cDNA and different proteins, such as or not show growth at the The interaction the of MIK and ID-MARK was using GST ID-MARK an the most 143 amino acids of the MIK protein, it not bind to an GST The partial MIK cDNA obtained from the yeast two-hybrid screen were used to screen maize embryo cDNA and genomic DNA The of corresponding cDNA and genomic allowed us to the MIK sequence MIK an kinase domain with high sequence to kinases of the GCK subfamily of family of Moreover, this domain the sequence which the motif of a of I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). a the kinase domain of MIK with of previously described GCK proteins. in a the and proteins described to S. A. M. 1999; PubMed Scopus Google Scholar). This of proteins the of proteins, which include the and as well as the proteins. The domain of is highly in sequence and is assumed to as a The domain of MIK does not show sequence with other GCK proteins and B. S. A. M. 1999; PubMed Scopus Google Scholar). the of we analyzed the expression of both Northern show that both MARK and MIK are and from after with a at in embryo and they are not in adult leaves also that both genes are from most adult the which are the that allow plants to generate their Antibodies against the intracellular domain of MARK, and against the regulator domain of MIK, were used to immunolocalization that both MARK and MIK in 15-DAP immature MARK strongly in the of the and in the of the which are the that are most in and in 15-DAP immature MARK also in the as a from to with the and activity of this at this of Although the in MIK are it also in the and of the immature the MARK a of most MIK to in the of the cells. MARK and MIK are with a of expression and are probably to of MARK and sections of 15-DAP immature embryos at the of the or the and incubated with or The and are shown by show the sections at MARK with MIK in and MARK and MIK interact in and are components of high complexes, we protein from 15-DAP maize immature embryos by MARK and MIK on a with at suggesting that they in We could not both proteins from probably the antibodies used were against the domains of both proteins and were with the proteins for We MARK and MIK interact in in COS-7 with an construct and a antibody against and MIK was also in with this antibody that MIK interacts with ID-MARK in of MIK activity by interaction in MARK and MIK interact in COS-7 cells. COS-7 were with corresponding to the proteins. of the of the and of the not were subjected to with antibodies against MARK and MIK activation of MIK phosphorylation activity. COS-7 were with corresponding to the proteins. to or were for phosphorylation activity on were by and by The of MIK and MARK proteins in the was by with and We have also used the COS-7 cell expression to MIK phosphorylation activity. and proteins were into COS-7 or in and or proteins were and used for phosphorylation does not or that ID-MARK is a phosphorylated in the Interestingly, the of a of phosphorylation the of a construct We report the cloning and characterization of two new plant genes coding for an atypical and a MAP kinase that could in signal transduction in MARK is an atypical with amino acid substitutions in its kinase-like domain that are to other kinases. results show the intracellular domain of MARK does not phosphorylate in vitro. We performed to the atypical present in III, and of MARK by the but the MARK protein not or This that other of amino acids for kinase activity have MARK This is also the case of the mammalian ErbB3 atypical which does not phosphorylate after the atypical have been S.A. EMBO J. 1994; PubMed Scopus Google Scholar). results that MARK is a that signals by Although plant protein with the sequence of an atypical has been described to protein C. J. J. Plant Mol. Biol. 23: PubMed Scopus Google Scholar), the a of and with these (see suggesting that phosphorylation-independent by atypical are in signal transduction in as they have been shown to in animal the last an of PRKs have been described in Although they are assumed to transduce signals by mechanisms to their animal V. J.M. T. Trends Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar), a of putative downstream signaling proteins with them have been described J.M. Plant 1998; PubMed Scopus Google Scholar, S.L. 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As ID-MARK interacts with the domain of MIK, we that interaction results in the of a of MIK kinase activity by this results a new mechanism by which atypical RPKs can transduce signals and could the of a still unknown signaling with in We are to Dr. Werr for the yeast two-hybrid maize embryo cDNA library and to and for the We for with the and Dr. for with the confocal We also and for and of the