A Single Pair of Acidic Residues in the Kinase Major Groove Mediates Strong Substrate Preference for P-2 or P-5 Arginine in the AGC, CAMK, and STE Kinase Families

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-θ and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release. Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-θ and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release. The term “basophilic kinase” has been used to describe kinases that preferentially phosphorylate substrates having basic residues in close proximity to the phosphorylation site (1Pinna L.A. Ruzzene M. Biochim. Biophys. Acta. 1996; 1314: 191-225Crossref PubMed Scopus (410) Google Scholar). Studies of PKA 3The abbreviations used are:PKAprotein kinase AAGCPKA, PKG, and PKC family kinasesCAMKcalmodulin-dependent protein kinaseGEL+1residue at position +1 in kinase GEL motif (see supplemental Fig. S1)STESte20 kinasesPAK1-LLPAK1.H83L.H86LPEN+1residue at position +1 in kinase PEN motif (see supplemental Fig. S1)YEM+1residue at position +1 in kinase YEM motif (see supplemental Fig. S1)MOPS4-morpholinepropanesulfonic acidMAPKKmitogen-activate protein kinase kinaseMAPKKKMAPKK kinasePAKp21-activated kinase in the late 1970s provided the first paradigm and showed a simple and powerful preference for Arg at P-3 and P-2 (2Kemp B.E. Bylund D.B. Huang T.S. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3448-3452Crossref PubMed Scopus (155) Google Scholar, 3Kemp B.E. Graves D.J. Benjamini E. Krebs E.G. J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google Scholar) (where P-n indicates the amino acid n residues N-terminal to the phosphorylated residue and P+n indicates the amino acid n residues C-terminal to the phosphorylated residue). PKC specificity proved to be more complex and to include preference for basic residues distributed on both sides of the phosphorylation site (4O'Brian C.A. Lawrence D.S. Kaiser E.T. Weinstein I.B. Biochem. Biophys. Res. Commun. 1984; 124: 296-302Crossref PubMed Scopus (62) Google Scholar, 5Turner R.S. Kemp B.E. Su H.D. Kuo J.F. J. Biol. Chem. 1985; 260: 11503-11507Abstract Full Text PDF PubMed Google Scholar, 6House C. Wettenhall R.E. Kemp B.E. J. Biol. Chem. 1987; 262: 772-777Abstract Full Text PDF PubMed Google Scholar). By 1990, some information on peptide specificity was available for about 20 kinases, of which the majority had evident preference for basic residues (7Kemp B.E. Pearson R.B. Trends Biochem. Sci. 1990; 15: 342-346Abstract Full Text PDF PubMed Scopus (807) Google Scholar). protein kinase A PKA, PKG, and PKC family kinases calmodulin-dependent protein kinase residue at position +1 in kinase GEL motif (see supplemental Fig. S1) Ste20 kinases PAK1.H83L.H86L residue at position +1 in kinase PEN motif (see supplemental Fig. S1) residue at position +1 in kinase YEM motif (see supplemental Fig. S1) 4-morpholinepropanesulfonic acid mitogen-activate protein kinase kinase MAPKK kinase p21-activated kinase The structural basis for the well defined basophilic preference of PKA was solved concurrently by two different approaches: mutational analysis (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar) and x-ray crystallography (9Yaffe M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (465) Google Scholar). The strong preference of PKA for Arg at P-3 was assigned to a single acidic residue (Glu-127) by both approaches. We refer to this residue as GEL+1 4The acidic pair pattern (yy, yn, ny, and nn) describes whether a kinase domain has an acidic residue at PEN+1 (first yes or no) and at YEM+1 (second y or n). to provide a general nomenclature rather than kinase-specific numbering. GEL refers to the consensus sequence of a conserved motif, and the +1 refers to its position relative to the first residue in that motif (supplementary Fig. S1). The GEL+1 acidic residue is located on the hinge that connects the kinase N-lobe to the GEL+1 both with and Arg at the P-3 position Biochim. Biophys. Acta. PubMed Scopus Google Scholar). in PKA has to be a general for mutational and the critical of this acidic residue in PKA as well as kinases T. C. K. T. A. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). is so general for basophilic kinases that degenerate peptide Arg as P-3 as the residue for C. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The strong preference of PKA for Arg P-2 was to be by two acidic PEN+1 and YEM+1 (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar, M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (465) Google Scholar). residues in the sequence the and but in three close to each in the PKA, in a has been to the to the of Arg at the P-2 position for Liu Y. J. Full Text Full Text PDF PubMed Scopus Google Scholar). it is general this to is in Arg preference at P-2 basophilic kinases, and is also complex between kinases in the residues at PEN+1 and YEM+1 (see and The between is not additional solved with Arg in substrate at and virtually mutational analysis has been of PEN+1 or YEM+1 residues. and has a new to this pair of residues. The of the AGC family kinase that the P-5 Arg in a substrate the by acidic residues at PEN+1 and YEM+1 A. S. J. PubMed Scopus Google Scholar). this for P-2 or P-5 Arg is to more the basophilic kinases, preference for Arg at positions than and the structural basis for this preference. is of both and it is to which basophilic kinases in specificity and to be in a analysis understanding the basis of peptide specificity and its We this analysis of 15 kinases the and kinase families using a new degenerate peptide library that preference for Arg at each substrate that PEN+1 and YEM+1 a in conferring strong P-2 or P-5 Arg preference in three kinase in the PAK family to be in exceptionally strong on P-2 Arg recognition mediated by two acidic residues. and single sequence and degenerate were as C-terminal on using as (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar). 15 kinases were PKA, PKG, and were and were and were A to residues of was in by and the protein was by at its with was in and by PAK1 and were the and in and using was the by and were to the phosphorylation of the of PAK1 was by using specific kinases in were in and on to The were with the and with the the were phosphorylated by in kinase as (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar). in phosphorylation in the of was in of degenerate of phosphorylation was to of substrate was to and of the preference for each position as on by and (9Yaffe M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (465) Google Scholar) for analysis of phosphorylation of degenerate We and in phosphorylation both of and of (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar). the preference for a position as is a by the of phosphorylation of the degenerate with Arg at that position with phosphorylation of degenerate of as for each of the were to each of that an Arg at a position the of the was 3) The preference for Arg at a position was as the of its in each of the for the P-3 position it is the of the peptide peptide peptide was the were for of for PKG, PKA, and the 1 20 for and the used in as well as and which were to kinase the 3) for the 1 8 and 1 which was Biochim. Biophys. Acta. PubMed Scopus Google for the and T. C. K. T. A. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google for PAK1 and the and Y. L.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google for the 20 1 1 and which was C. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google for the 20 1 and which was Liu Y. J. Full Text Full Text PDF PubMed Scopus Google and for the is 20 and to the and was by the of the was in to a which were A is in kinase domain has sequence to and is in kinase domain has to provided by J. of and were first to a and or were kinase to and a was to the kinases to of substrate peptide to were and the were at for and were by that strong preference for Arg at P-2 or P-5 in to preference for Arg at is of basophilic kinases (1Pinna L.A. Ruzzene M. Biochim. Biophys. Acta. 1996; 1314: 191-225Crossref PubMed Scopus (410) Google Scholar). it be to whether P-2 and P-5 uniquely the We this in an We the pattern of basophilic preference kinases by determining the of Arg or in phosphorylation Fig. analysis of a of basic preference. P-2 and P-5 the only two positions to at which the of Arg is than the Arg in the We analysis of the structural basis of this pattern of basophilic preference. A degenerate peptide library was to the preference of a kinase for is on a of degenerate each having Arg and 1 Fig. The positions of the Arg residues the so that it of two Arg position to The positions with a degenerate of residues. Arg was rather than it has been that the for Arg not K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar, A. S. J. PubMed Scopus Google Scholar, and S. Cantley L.C. Biol. Full Text Full Text PDF PubMed Scopus Google and supplemental Fig. The of Arg than a single Arg for two to be to whether were strong of of the of phosphorylation of degenerate with single to of this was by analysis of three kinases peptide specificity and Arg preference has been by approaches: PKA, and with kinase on phosphorylation of the Fig. the to a peptide with Arg at a position as of Arg preference at a position is by phosphorylation of with the of the The of this is a for each residue position that the relative preference or for an Arg at that position with Arg at positions in the peptide The the analysis with about the three PKA was in the 1970s to a simple and strong pattern of Arg preference at P-2 and P-3 B.E. Graves D.J. Benjamini E. Krebs E.G. J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google which is evident with the The peptide with Arg at P-2 and P-3 is uniquely well phosphorylated Fig. and the strong preference at P-2 and P-3 PKC is known to PKA in having a more distributed preference for Arg not only at P-2 and P-3 but also for Arg at C-terminal C. Wettenhall R.E. Kemp B.E. J. Biol. Chem. 1987; 262: 772-777Abstract Full Text PDF PubMed Google Scholar, A. S. J. PubMed Scopus Google Scholar). preference is in the relative phosphorylation of Fig. this the in the Arg at and is known to an Arg preference at P-3 and P-5 PubMed Scopus Google which is evident both in Fig. and in additional AGC kinases were PKG, known to be to PKA in Arg showed strong Arg preference at P-2 and P-3 and preference at P-5. and each with the three positions of Arg and the relative of the positions between and the P-2 position was by the Arg preference. The strong preference of for Arg at P-2 was in Fig. and the AGC kinases PKA, PKG, and for which the preference was at P-3. Arg preference has not been in AGC kinases in lacking strong Arg preference at P-2 or analysis of A, relative phosphorylation by of a of degenerate with at but of a The general of each peptide in the is each a degenerate position and a A at the is also in each peptide for on of the has an Arg at a different position to on the as position of Arg preference for and three as preference for Arg at the The of analysis on the that the position of a strong Arg preference be of residues in a We were to this for it a residue as or at the position (8Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (83) Google Scholar, K. A. Cantley L.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We an additional of degenerate of which had a at the The also had a single Arg position between and Analysis of phosphorylation of this by confirmed that the P-2 position was the single position for The two were and P-3 the relative of Arg as by the analysis was confirmed by the analysis using the with at We used mutational analysis to whether the strong P-2 Arg preference of had the structural basis as the P-2 Arg preference of residues and were each to a residue at the positions of to and to of the of demonstrated that the had not the of the kinase on a substrate with basic residues at positions Fig. Arg of the kinases were using the protein showed the P-2 Arg preference with the kinase The protein had greatly preference for Arg at The protein also had a in preference for Arg at additional protein as a residue was is the only acidic residue is the protein showed of P-2 Arg preference. the P-2 preference of is by and The with the PKA that strong P-2 Arg preference on of the pair of acidic residues in the PEN+1 and YEM+1. We conservation of residues AGC kinases and the two kinase to the AGC and is to describe a kinase by a two acidic pair pattern (yy, yn, ny, or nn) whether the kinase domain has an acidic residue at PEN+1 (first yes or no) and at YEM+1 (second y or n). The two to this acidic pair the and in acidic pair Most of the AGC kinases a pattern acidic residues at both Most of the a pattern acidic residue at PEN+1 but not at and of the kinases an pattern acidic residue at YEM+1 but not at each of kinases which the of of acidic residues and of acidic residues in kinase pair residue residue family of kinases in of kinases in family with pattern and at GEL+1 in a new analysis AGC family kinases 1 and the only with a strong P-2 Arg preference. the with a but had a strong P-2 Arg preference. The was which showed a strong P-5 Arg preference of a P-2 Arg preference. for the that analysis of J. Nat. Biol. PubMed Scopus Google that the pair of acidic residues in an which strong preference for Arg at P-5 a residue to that (see acidic residues at the PEN+1 and YEM+1 positions both for strong P-2 or P-5 Arg kinases not an acidic residue at acidic pair is by of peptide specificity on at of kinases been to strong Arg preference at and been to strong P-2 Arg preference K. A. Cantley L.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, R.B. Kemp B.E. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar, C. C. Graves D.J. J. T. Cantley L.C. Biol. 1996; PubMed Scopus Google Scholar, M. Lawrence D.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. E. PubMed Scopus Google Scholar, Biochem. J. PubMed Scopus Google Scholar, T. L. Chen L. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. 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PubMed Scopus Google Scholar). with this of kinase with an acidic pair demonstrated Arg preference P-3 in the family the kinases with a acidic pair of kinases and to the PAK and three and to the PAK Analysis of PAK1 and demonstrated that both had an exceptionally strong preference for Arg at P-2 the basophilic kinases the had preference for Arg at with of an acidic residue at which is in to the P-2 Arg preference of the The three kinases with a acidic pair pattern to a of the family and Analysis of of the MAPKK kinases, showed that its Arg preference was at but that preference was so that its is that residues in the kinase domain the PEN+1 and YEM+1 acidic residues in strong P-2 Arg We structural by a of with The that the of located on the in close proximity to which position it to the acidic for Arg that basic residue is conserved in and the of the PAK the only kinases of the family that a residue pattern analysis a strong P-2 or P-3 Arg preference. The preference of for Arg at P-2 is with its acidic pair We whether and its preference for Arg at this analysis that is an of PAK E. Huang Chen T. L. Biol. PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (62) Google Scholar). residues a critical residue in the analysis of amino sequence the site in PAK family that Arg at the P-2 position relative to the site is P-2 is the of strong Arg preference of We that of two residues (PEN+1 and the were in had in with in of phosphorylation was with and PAK1 protein and had of that activity be on of the substrates had to be for phosphorylation by PAK1 was not to of of the to not kinase activity not The the strength of Arg preference at the P-2 and P-5 positions basophilic kinases and the structural basis for that preference. The on the of the binding for P-2 Arg in the of the kinase of the for P-5 Arg binding by some and 3) of PAK family on P-2 Arg The that acidic PEN+1 and YEM+1 residues that were first to strong P-2 Arg preference for PKA also the of strong P-2 Arg preference for kinases of the AGC and pair of residues is in AGC kinases The of an additional AGC the general of the binding in the of the kinase S. S. K. L. C. L. L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that the PEN+1 and YEM+1 residues mediate the strong P-2 Arg preference of not be is in the kinase as the PAK family of kinases is more to PKA, and kinase only the general of the binding and the two acidic residues to mediate strong P-2 Arg preference also with the by and J. Nat. Biol. 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Nat. 2004; PubMed Scopus Google Scholar). confirmed that has Arg preference and acidic pair and strong P-5 Arg preference. kinases lacking the acidic pair pattern been to strong P-5 Arg preference. is that the PEN+1 and YEM+1 residues the in protein kinases of P-5 Arg recognition in the PEN+1 and YEM+1 acidic residues to strong specificity for P-2 or P-5 Arg of two residues was evident in PKA as assessed both in the and by a more general of two residues in analysis of sequence conservation and Arg preference for P-2 or P-5 indicates that strong preference is almost in kinases with the acidic pair pattern and only in and which an acidic residue at YEM+1 P-2 Arg preference. mutational analysis of both and PAK that both residues greatly to this strong Arg preference. is that of and PAK at the YEM+1 residue a in P-2 Arg preference than of the PEN+1 is the YEM+1 residue has been the YEM+1 residue is not in the of kinase domain residues used by Proc. Natl. Acad. Sci. U. S. A. 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The strength of P-2 Arg which is to also be by the of residues the the of this is which has a P-2 Arg preference of this pair as is to a basic residue on binding by the acidic A more of this is evident in between Arg P-2 preference is in than and 1 and kinase and virtually conservation in the not is that this in or relative between the residue The specificity of PAK is as on the of and C.A. PubMed Scopus Google Scholar). PAK as a basophilic kinase basophilic kinases is not by the PAK specificity for Arg at P-2 basophilic kinases in the PAK and PAK not a strong Arg preference at which virtually known basophilic a in kinases, which the GEL+1 acidic residue that is almost used by AGC and to a P-3 Arg preference. on P-2 Arg recognition is a for PAK phosphorylation of of having of Arg in the of PAK1 and each phosphorylated of the were at as well phosphorylated as the peptide of phosphorylated had Arg at but only of the more phosphorylated perfect of phosphorylation with Arg at P-2 was not with of the kinases not is with the of activity in PAK1 at PEN+1 and YEM+1. PAK is on Arg at which strong in PAK phosphorylation kinases the families of in G. D.B. T. S. PubMed Scopus Google Scholar). protein mediate the protein kinase domain has been by the in the that has been used by evolution to mediate phosphorylation S. J. Biol. PubMed Scopus Google Scholar). The a for two acidic PEN+1 and in the of the residues been used by evolution to strong preference for basic residue at the P-2 or P-5 position in The PAK family a of this in which P-2 Arg with this pair of residues is for kinase We for of for for and the for with