Structural and Functional Insights into the Regulation of Arabidopsis AGC VIIIa Kinases

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta. The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta. Postembryonic plant development is coordinated through the integration of environmental signals with those derived from intrinsic plant growth regulators. Because the plant cell wall inhibits cell motility and prevents direct cell-to-cell contact, the transmission of information between cells relies largely on the activation of an impressive array of intercellular signaling machinery. The completed sequence of the model organism Arabidopsis has revealed that roughly 10% of the genome is devoted to signaling-related genes (1The Arabidopsis Genome InitiativeNature. 2000; 408: 796-815Crossref PubMed Scopus (7085) Google Scholar). Included in this sector are the more than 1,000 annotated genes predicted to encode serine-threonine protein kinases, nearly double the number found in the genomes of representative metazoan organisms (2Champion A. Kreis M. Mockaitis K. Picaud A. Henry Y. Funct. Integr. Genomics. 2004; 4: 163-187Crossref PubMed Scopus (91) Google Scholar). One mechanism for the observed expansion of kinases into larger gene families in plants as compared with animals or fungi is the amplification of low copy number genes into larger gene families, which may perform divergent or stage- and tissuespecific functions (3Force A. Lynch M. Pickett F.B. Amores A. Yan Y.-L. Postlethwait J. Genetics. 1999; 151: 1531-1545Crossref PubMed Google Scholar). A major challenge of post-genomic Arabidopsis research will be to understand the extent to which duplicated genes retain ancestral function or are conscripted by other signaling networks to regulate disparate cellular processes. One example of a gene family that has undergone expansion in plants, is the AGC family of serine-threonine protein kinases. In yeast and mammals, these kinases have been implicated in the regulation of transcription, apoptosis, cell proliferation, insulin signaling, and cytoskeletal remodeling (4Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (601) Google Scholar, 5Dong L.Q. Landa L.R. Wick M.J. Zhu L. Mukai H. Ono Y. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5089-5094Crossref PubMed Scopus (87) Google Scholar, 6Nicholson K.M. Anderson N.G. Cellular Signaling. 2002; 14: 381-395Crossref PubMed Scopus (1372) Google Scholar, 7Suzuki E. Hanada K. Toledo M.S. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14788-14793Crossref PubMed Scopus (73) Google Scholar, 8Watson R.T. Kanzaki M. Pessin J.E. Endocrine Rev. 2004; 25: 177-204Crossref PubMed Scopus (352) Google Scholar). Despite the breadth of cellular processes controlled by these proteins, numerous studies suggest that phosphorylation by 3-phosphoinositide-dependent kinase 1 (PDK1), 4The abbreviations used are: PDK1, 3-phosphoinositide-dependent kinase; PID, PINOID; PIF, PDK1-interacting fragment; GST, glutathione S-transferase; His, polyhistidine; GFP, green fluorescent protein; PID-ID, PINOID insertion domain; MBP, myelin basic protein; HRP, horseradish peroxidase.4The abbreviations used are: PDK1, 3-phosphoinositide-dependent kinase; PID, PINOID; PIF, PDK1-interacting fragment; GST, glutathione S-transferase; His, polyhistidine; GFP, green fluorescent protein; PID-ID, PINOID insertion domain; MBP, myelin basic protein; HRP, horseradish peroxidase. itself an AGC kinase, is an evolutionarily conserved mechanism for regulation of AGC kinase activity (9Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (661) Google Scholar). In contrast to the detailed information available for their yeast and metazoan counterparts, the signaling processes associated with AGC kinase activity in plants, and the mechanisms by which kinase activity is regulated, remain poorly characterized. The 39 identified Arabidopsis AGC protein kinases can be phylogenetically grouped into five distinct subfamilies (10Bögre L. Okresz L. Henriques R. Anthony R.G. Trends Plant Sci. 2003; 8: 424-431Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The AGCVI subfamily includes two S6K homologues that have been shown to respond to regulation by phytohormones (11Turck F. Zilbermann F. Kozma S.C. Thomas G. Nagy F. Plant Physiol. 2004; 134: 1527-1535Crossref PubMed Scopus (90) Google Scholar). The AGCVII class contains eight uncharacterized homologues of the nuclear Dbf2-related kinases (12Tamaskovic R. Bichsel S. Hemmings B.A. FEBS Lett. 2003; 546: PubMed Scopus (91) Google Scholar). The AGC subfamily of genes to which is in the of growth Y. K. Plant J. 2002; PubMed Scopus Google Scholar). The two PDK1 homologues of Arabidopsis in a The and kinase class is subfamily which contains The kinases a subfamily identified by two a conserved implicated in the of and the of a of sequence between and J. PubMed Scopus Google Scholar). (10Bögre L. Okresz L. Henriques R. Anthony R.G. Trends Plant Sci. 2003; 8: 424-431Abstract Full Text Full Text PDF PubMed Scopus (154) Google have the AGC subfamily into two and and AGC kinases to have been and in The and domains that respond to and a that the kinase domain M. M. K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Trends Plant Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The kinase, identified through its interaction with PDK1, is in growth and is in to and R.G. Henriques R. A. G. M. L. J. 2004; PubMed Scopus Google Scholar, D. F. L. H. H. S.C. H. 2004; PubMed Scopus Google Scholar). the PINOID has been identified and was shown to a fundamental role in the asymmetrical localization of auxin proteins the cell membrane J. G. D.R. Plant J. 8: Scopus Google Scholar, J. D. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. D. R. PubMed Google Scholar, J. M. D. A. R. K. G. K. R. 2004; PubMed Scopus Google Scholar). A AGCVIIIa kinase, was in a yeast for proteins that interact with the M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google the of this interaction is two other kinases of this and were identified in Arabidopsis on their to the gene and have been shown to regulate in to Plant J. PubMed Scopus Google Scholar). the of PID, have to mutant associated with of AGCVIIIa gene of the 16 genes encoding AGCVIIIa kinases have been identified as plant corresponding to the insertion not have The for these is and the majority of these proteins are to or functional a elucidating those processes that are controlled by these kinases, we have the characterization of the AGCVIIIa subfamily in we have gene expression and localization of the corresponding AGC by was with Plant of plant to the the was for 1 with and the of was by amplification the that were used in the of were the was used as for the in the of of to the the were and of the was used as a was in 1 of in the of and for the were to the to the amplification were for for 1 and for were on a sequences used in this are available and of in was from other were by amplification derived from plant or on the and domain of expression of the AGC gene the used to the are available were and into the corresponding of to glutathione PDK1 was as H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and were into and the The domain of PID was into and by a domain on the PINOID were and into the and were into expression was with 1 the were for and the were by cells proteins were in 1 and and on for was to and the was by for in an The was with and of and the was for The was with and was as for the protein that was used in of or AGC proteins to were with of for with The were in and two in with and and the proteins were in by the proteins were to membrane and the PDK1 was by with an and an The was with analysis of AGC by of to was with kinase 1 in of and and for were by of and the were on the were for 1 in with with 10% acid, and for by AGC kinase of myelin basic protein and of were to the phosphorylation and the was for were on The corresponding to was from the and was by and of GFP-tagged AGC yeast the was by a the and from S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google into the and to was by into a the encoding PID, and were into with and into domain between PID and the PID sequence between the and was with a the corresponding sequence in was by the PID between and by The were into the of with The PID insertion domain was and into were used to Saccharomyces cerevisiae on the as H. Y. A. J. Scholar). were that for in or yeast were to by for and in expression in tobacco BY-2 and were from the corresponding with and and into the and of the the The was by the the gene from the into were into by were with in with and of BY-2 tobacco cell were with of of and the cells were with the in a in the cells were with to and by for cells were by a and a was on a were with a and for yeast and tobacco of PID to was protein-lipid as S. Alessi D.R. J. 1999; PubMed Scopus Google Scholar). with were in a of in for PID protein was as H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and to the The were with with the were with by an The was with analysis The was to of the Arabidopsis AGCVIIIa AGCVIIIa kinase subfamily is of predicted proteins that are located the Arabidopsis genome The kinases are grouped phylogenetically by sequence and the of domains. In to the to that is with the group J. PubMed Scopus Google these kinases are identified by the of an insertion domain located between and domain sequence between family and in from to of the AGCVIIIa insertion domains. of the insertion domains five in conserved in more than of the AGCVIIIa and are those to more than of the members of AGCVIIIa basic The domain found in is were the of sequences to the majority of the AGCVIIIa kinases are a of of PDK1 substrates. The of these sequences is a C-terminal domain the PDK1-interacting or In phosphorylation of the the is to be required for PDK1 (9Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (661) Google Scholar). of annotated Arabidopsis AGCVIIIa kinases a C-terminal but not to the found in A. M. Alessi D.R. J. 2000; PubMed Scopus Google Scholar). contains the sequence the remaining proteins, and not an C-terminal domain In yeast and PDK1 its substrates on the a conserved activation loop to S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A nearly sequence found in the plant AGCVIIIa kinases, from the PDK1 in the of for the kinases, and a in which the 1 is to the conserved kinase which an of family the insertion domains found in AGCVIIIa kinases are in that the insertion domains group into five with of sequence the of and the kinases a in basic the C-terminal of the domain A single kinase, contains a domain of the basic between is the insertion domain sequences a are conserved between Arabidopsis AGCVIIIa kinases and proteins in and plant not of the insertion domains by evolutionarily and of sequences plants that these are to be for kinase functional or between PID and the 16 homologues predicted by the annotated Arabidopsis we have corresponding to of the We were to a for analysis that of this gene is to M. L. Plant Physiol. 2004; PubMed Scopus Google and to the that may be expressed low or in a number of cells the In PDK1 and of AGCVIIIa phosphorylation of numerous AGC kinases by the PDK1 that and activation of substrate kinases (9Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (661) Google Scholar). In plants, information the role of PDK1 as a of AGC kinase function is PINOID has been shown to be by PDK1 in and in H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). other Arabidopsis AGC kinases, and were identified as PDK1 in a yeast R.G. Henriques R. A. G. M. L. J. 2004; PubMed Scopus Google Scholar). A single kinase, an of plant cell has been shown to be a substrate for the PDK1 kinase J. 25: PubMed Scopus Google Scholar). PDK1 is a of other Arabidopsis AGCVIIIa kinases, we the of these kinases to as substrates for PDK1 in kinases to and cell PDK1 by analysis that of the kinases interact with PDK1 in A and PDK1 was not in The that and which an to PDK1 is with in which of the PID domain to PDK1 and interaction of PDK1 with in domain J. 25: PubMed Scopus Google Scholar). that the domain of the AGC kinases is not required for PDK1 in We in phosphorylation of the AGCVIIIa proteins to PDK1 shown in the majority of the kinases a in phosphorylation with PDK1, its Arabidopsis PDK1 with AGC kinases. and which the domain and the activation loop 1 are not by PDK1, the that proteins to PDK1 in phosphorylation of which the C-terminal was by with PDK1 of the sequence revealed a sequence of the protein is that this functions as a and may the in phosphorylation in to which contains a C-terminal domain and a sequence the activation loop was not by PDK1 in is the AGCVIIIa kinase of the of an the that this domain may regulatory sequences that PDK1 or we the an were the or that this not have in regulatory function not demonstrate that the domain to be a for regulation of the AGCVIIIa kinases by PDK1 (9Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (661) Google Scholar, H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google sequences be required to PDK1 phosphorylation of substrates. PDK1 phosphorylation has been shown to activation of substrate kinases. the in phosphorylation in to PDK1 observed for the majority of the AGCVIIIa kinases is to from direct phosphorylation by PDK1 and in the phosphorylation of kinases with in kinase we the of kinases, and that show phosphorylation with PDK1, and which is not by PDK1, to the substrate shown in phosphorylation of and by PDK1 in in of with PDK1 on its that Arabidopsis PDK1 is to be an but not of AGCVIIIa kinase of and the domain and an activation loop the 1 are present in AGCVIIIa kinases that are by the of these to PDK1 interaction and AGC kinase we and in into the kinase, which is not an in substrate for We the of a domain was sufficient to into a substrate for the of a C-terminal domain to and kinase in the of PDK1 1 with and of with PDK1 in a in protein as compared with the protein The observed in phosphorylation in to PDK1 was in a in the activity of substrate of a domain in an in the mechanism of activation to be largely of PDK1 We of the activation loop to or in with the in phosphorylation of the protein by phosphorylation of the of PDK1 or the domain 1 and with and and and with and in which the conserved activation loop of PID was by in a but in phosphorylation as compared with and PDK1 suggest that the conserved kinase is to function in PDK1 phosphorylation that the domain the activation loop or are sufficient to PDK1 substrate The AGCVIIIa used analysis of from and to the expression of AGCVIIIa kinases. Consistent with to the we were to a for the gene in of the not The other kinases a of expression the of and of the kinases to be expressed in In and which group in protein and insertion sequence were expressed in The gene was expressed in and In was expressed in the and gene and a nearly expression with genes expressed in with the of and expression in the and which are phylogenetically expression domains. was expressed in and expression was to and The and a divergent expression was expressed in expression to be to and in expression was observed in the was expressed in expression was from and In expression was to and to a extent to expression was not observed in of the Consistent with its PID expression was found in and with in other of Arabidopsis AGCVIIIa AGCVIIIa kinases are the we the localization of and PID to Because expression of regulatory proteins in plants in low protein cell and and the of and we kinase localization by in S. of in yeast of expression revealed that localization from that observed for the A and and expression were nuclear and The nuclear localization of in yeast is to that observed expression of GFP-tagged and in the protein was preferentially localized to the cell periphery in a to that observed in expression of PID the of the Plant PubMed Scopus Google Scholar). PID expression was observed suggesting that PID may in or more the expression may from protein that the observed localization protein interactions as to with yeast cellular we tobacco BY-2 cell with and The localization were to those observed in yeast that and PID are preferentially localized to the cytoplasm and cell and We the observed localization of PID on activation by A mutant was expressed in In this mutant the two activation loop required for PDK1 activation of PID H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were by The which has low of a localization to that of the protein that phosphorylation by PDK1 is not required for protein localization. The PINOID which protein domains to in we which to the cell and expressed PID the insertion domain and of the conserved domain was with the corresponding of of this in yeast and tobacco BY-2 cells in localization of the protein A and of the in which the PID insertion domain was to the and of in a peripheral localization to that of and the insertion domain was sufficient to direct protein the we yeast with an insertion domain to The localization of the insertion domain was from that of the that sequences outside of this are not for protein localization. The peripheral localization of PID and the that these proteins might or may directly to We used to PID with We find that PID binds but not to and to phosphatidic that the observed peripheral localization of PID the cell may through direct plasma membrane The AGC kinases a conserved class of regulatory proteins that are present in eukaryotic of of the plant AGC and PDK1 to be present in the of PDK1 and to the associated with the corresponding yeast that the signaling processes by these kinases are and evolutionarily conserved A. J. Alessi D.R. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the distinct AGCVIIIa kinases a kinase subfamily functional characterization has been by the of single mutant In this we have the regulation of the Arabidopsis AGCVIIIa kinase subfamily as a in protein The domain of the Arabidopsis AGCVIIIa kinases the domain found in the of phosphorylation and of and PDK1 have shown that the interaction of these proteins is in through of the domain a located in the of PDK1 A. M. Alessi D.R. J. 2000; PubMed Scopus Google Scholar, Komander D. Thomas M. Alessi D.R. Aalten D.M. J. 2002; PubMed Scopus Google Scholar). through the interaction of the domain with the have been to function in substrate protein and in the of through substrate of PDK1 A. M. Alessi D.R. J. 2000; PubMed Scopus Google Scholar). have been identified in AGC kinases and analysis that to PDK1 the domains of substrate kinases with their to the protein in an in show that of the AGCVIIIa kinases and which the were to interact with are in with in which of conserved the domains of PID and in in protein on protein H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. 25: PubMed Scopus Google Scholar). Anthony R.G. Henriques R. A. G. M. L. J. 2004; PubMed Scopus Google used yeast analysis to demonstrate that an domain is for the interaction of with PDK1 in these that sequences outside of the domain a role in interactions in and may to protein in the of a domain to be a for functional interactions between PDK1 and plant AGCVIIIa kinases. of evidence that sequences outside of the domain to substrate which contains a is not by PDK1 in the of a domain to in an in kinase with PDK1 kinase suggest that the domain is not sufficient to PDK1 One for the PDK1 activation of is that may retain an ancestral that and binds the In this with PDK1 in activation through for from but not from plants R.G. Henriques R. A. G. M. L. J. 2004; PubMed Scopus Google as an in substrate for PDK1, that are to the of PDK1 to regulate AGCVIIIa activity in The of a domain in and correlates with the of a conserved in the activation loop to We this PDK1 by a corresponding into the PID of this not PID of for the in or on PDK1 that the remaining Arabidopsis AGCVIIIa kinases are conserved in this that sequences that PDK1 substrate are to the or C-terminal or the kinase domain Because the majority of AGCVIIIa kinases to interact with PDK1, that mechanisms in to kinase activation substrate this we as a of functional between kinases. that numerous AGCVIIIa genes and or expression to a number of and these encode conserved the corresponding regulatory sequences to have undergone AGCVIIIa genes were found to be expressed in we the that expression a is to a cell or the expression domains may in the of associated with these mechanism to the functional of kinases, as PDK1, with is through localization of substrates to distinct PubMed Scopus Google Scholar). The localization of Arabidopsis AGCVIIIa kinases to and and the cell periphery in yeast that kinases are to through interaction with distinct cellular has been that interaction with PDK1 might PID to the plasma membrane H. Anthony R.G. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). from the present this model into nearly of the Arabidopsis AGCVIIIa kinases interact with PDK1 in but PID has been to for the plasma membrane or plasma the of a of activation loop shown to be phosphorylation for PDK1 not protein localization. the of PID for phosphoinositides the that membrane localization may be an intrinsic of the The suggest that activation and localization of PID are is in contrast to the of and Plant PubMed Scopus Google that expression of a PID protein the peripheral localization of PID expressed in Arabidopsis is that in the of the the of which of the of the insertion with might in a that prevents interaction between the insertion domain and its localized in protein localization associated with the of insertion domains between PID and and the of the insertion domain to evidence that this protein domain is sufficient to PID to the cell the metazoan AGC kinases, members of the which cell growth and a insertion a corresponding to that by the insertion domain of plant AGCVIIIa kinases (12Tamaskovic R. Bichsel S. Hemmings B.A. FEBS Lett. 2003; 546: PubMed Scopus (91) Google Scholar). domain a localization that kinases to the nucleus Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The in domain and function may that protein sequences in this for interaction with proteins or other of the cell to localization. The between yeast and tobacco expression that plant AGCVIIIa kinases to The observed protein are to the of plant kinases for distinct will be to the insertion domains of other AGC kinases that of PID, the of localization. The to in insertion domain with distinct localization will be an in the of or of the of and substrates will to be to AGCVIIIa kinases to signaling a in the roles of these regulatory proteins in plant We for The was a from J. of was from the Arabidopsis We and for and of the