proper signal transmission by protein kinases requires that they phosphorylate specific substrates at defined sites .
multiple mechanisms can act in concert to provide substrate specificity for kinases , including kinase - substrate colocalization , compartmentalization through the use of scaffold proteins , substrate recruitment through kinase adaptor subunits , and direct physical interactions between kinases and their substrates ( ubersax and ferrell , 2007 ) . in order for substrate phosphorylation to occur ,
the residue phosphorylated by the kinase must bind at least transiently to the catalytic cleft of the kinase .
accordingly , protein kinases tend to phosphorylate substrates in the context of consensus sequence motifs that have complementarity to the kinase active site ( pinna and ruzzene , 1996 ) .
such kinase phosphorylation site motifs play an important role in targeting kinases to specific substrates within the cell , as well as directing kinases to phosphorylate specific sites on their substrates .
one important aspect of the kinase phosphorylation site motif is the identity of the phosphorylation site residue itself .
almost all kinases in eukaryotes phosphorylate protein substrates on ser , thr , or tyr residues .
ser - thr kinases , which make up approximately 80% of the human kinome , comprise several groups phylogenetically distinct from tyr - specific kinases ( manning et  al . , 2002 ) .
accordingly , ser - thr kinases possess conserved signature residues within the catalytic domain important for accommodating a small , aliphatic phosphoacceptor residue at the active site ( taylor et  al . , 1995 ) .
likewise , a distinct set of conserved residues characterizes tyr kinases , which must accommodate a large , aromatic residue .
interestingly many , though not all , ser - thr kinases are significantly selective for either ser or thr as the phosphoacceptor residue ( pinna and ruzzene , 1996 ) .
for example , camp - dependent protein kinase ( pka ) strongly favors ser over thr in peptide substrates , and a large majority ( > 90% ) of established in  vivo phosphorylation sites are at ser residues ( aimes et  al . , 2000 ;
conversely , the kinase lkb1 activates numerous downstream protein kinase substrates by phosphorylation exclusively on thr residues ( lizcano et  al . , 2004 ) .
although substrate specificity studies have predominantly been conducted in  vitro , evidence is now emerging indicating that phosphoacceptor residue identity significantly influences substrate phosphorylation efficiency in living cells ( kang et  al . , 2013 ) .
thus , the presence of a preferred phosphoacceptor residue appears to be important for targeting of specific substrates by ser - thr kinases .
, we show that phosphoacceptor preference of ser - thr kinases is determined largely by the identity of a single residue , which we term
 this residue is located within the kinase activation segment , a conformationally flexible loop important for kinase regulation .
mutagenesis of this residue is sufficient to change the phosphoacceptor preference for kinases from distinct groups in a predictable manner .
identification of this residue therefore establishes a simple rule that can be used to predict phosphorylation site preference for a kinase of unknown specificity .
we go on to show by x - ray crystallography of kinase - peptide complexes that conformation , rather than binding affinity , drives phosphoacceptor preference .
overall , these studies explain how a protein kinase is able to discriminate between ser and thr , two residues that differ only by a single methyl group .
we recently analyzed the peptide substrate specificity of a large number of ser - thr kinases from s.  cerevisiae ( mok et  al . , 2010 ) .
among these kinases , 32 preferred ser , 10 preferred thr , and 14 lacked substantial phosphoacceptor residue preference ( table s1 available online ) . while about half of the nonselective kinases belonged to a single phylogenetic group ( the cdk- , mapk- , gsk3- , and ckii - related [ cmgc ] kinase group ) , possibly attributable to a distinct peptide - binding mode ( brinkworth et  al . , 2003 ;
, 1999 ; mok et  al . , 2010 ) , all other kinase groups included both ser- and thr - specific kinases ( table s1 and figure  s1 ) .
thus , in contrast to the clear distinction between tyr kinases and ser - thr kinases , ser - preferring and thr - preferring kinases can not be readily distinguished based on phylogeny .
residues within the kinase catalytic domain that determine target specificity covary with corresponding residues in the substrate peptide ( li et  al . , 2003 )
. computational analysis of our yeast kinase data identified residues that covaried between kinases and their consensus peptide sequences ( yip et  al . , 2011 ) .
one residue correlating strongly with phosphoacceptor preference is found immediately downstream of a conserved asp - phe - gly ( dfg ) sequence at the n terminus of the kinase activation loop ( hereafter referred to as the dfg+1 residue ) .
consistent with a potential role in controlling kinase specificity , this residue is located within the substrate - binding cleft ( goldsmith et  al . , 2007 ) .
among yeast kinases analyzed , ser - selective kinases had larger hydrophobic residues ( predominantly leu , phe , and met ) at this position , while most thr - specific kinases had the -branched aliphatic residue ile at this position .
for example , lkb1 and pdk1 , which phosphorylate primarily thr residues in substrates ( lizcano et  al . , 2004 ; mora et  al . ,
2004 ) , have the -branched residues val and thr , respectively , at the dfg+1 position .
in addition , human protein kinase c isozymes , which prefer ser as the phosphoacceptor ( ferrari et  al . , 1985 ; house et  al . , 1987
) , have the straight - chain aliphatic residue met at the analogous position . based on its location within the kinase fold and the strong correlation between its identity and kinase specificity , we hypothesized that the dfg+1 residue might be an important determinant of phosphoacceptor specificity for ser - thr kinases .
based on this hypothesis , mutation of the dfg+1 residue within ser - thr kinases should alter specificity in a predictable manner .
we initially focused on human kinases in the ste7- , ste11- , and ste20-related ( ste ) group .
most kinases in this group , including those in the mammalian ste20-like ( mst ) kinase family , are thr selective ( miller et  al . , 2008 ) , while members of the p21-activated kinase ( pak ) family are ser selective ( rennefahrt et  al . , 2007 ) .
phosphorylation site specificity within these families correlates with dfg+1 residue identity ( val for mst kinases and phe for paks ) .
we generated dfg+1 exchange mutants of these kinases ( mst4 and pak4 ) and examined their phosphorylation specificity alongside their wild - type ( wt ) counterparts using optimized synthetic peptides that differ only at the phosphosite .
as anticipated , wt mst4 preferentially phosphorylated the thr peptide , while wt pak4 preferentially phosphorylated the ser peptide ( figure  1a and table 1 ) .
furthermore , mutation of the dfg+1 residue was sufficient to invert phosphorylation site specificity for both kinases on both consensus peptide substrates and peptide libraries ( figures 1a and s2 ) .
notably , we found that for a given kinase , the km values for phosphorylation of the ser and thr peptides were similar , but phosphoacceptor residue identity had a large effect on kcat ( table 1 ) .
these results suggest that phosphoacceptor preference is not mediated by differences in substrate binding affinity . to determine whether the dfg+1 residue controls phosphoacceptor preferences among ser - thr kinases in general , we tested the effect of mutating this residue in two more distantly related kinases : snf1 ( the yeast ortholog of amp - activated protein kinase , from the ca / calmodulin - dependent protein kinase - related group ) and pka ( from the pka- , pkg- , and pkc - related [ agc ] group ) .
we generated a series of snf1 mutants in which the dfg+1 residue ( leu198 ) was changed to residues most commonly found in other kinases at this position and evaluated the ability of these mutants to phosphorylate ser- and thr - containing peptides ( figure  1b ) .
mutation of leu198 to a small or -branched residue ( ser , val , or ile ) converted it to a thr kinase , while mutation to phe in snf1 retained a preference for ser . unexpectedly ,
snf1 , predicted to be ser specific , did not discriminate between ser and thr .
for pka , mutation of the dfg+1 residue ( phe187 ) to val inverted its selectivity from ser to thr specific ( figure  1c ) . taken together ,
these data indicate that the dfg+1 residue plays a predominant role in dictating phosphoacceptor specificity across multiple kinase groups .
if the dfg+1 residue is the primary determinant of ser - thr kinase phosphoacceptor specificity , the identity of this residue should allow us to predict the preference of an uncharacterized kinase .
most kinases within the human agc group appear to prefer ser , and in keeping with this , leu , met , and phe are found most frequently at the dfg+1 position .
kinases in the rock family are outliers in that they have a -branched thr dfg+1 residue , suggesting they are likely to prefer thr over ser . using a pair of matched peptides differing only at the phosphoacceptor site
, we found that rock1 indeed had a significant ( approximately 2-fold ) preference for thr as the phosphoacceptor ( figure  1d ) . to assess the contribution of the dfg+1 residue to substrate phosphorylation in cells
, we examined the ability of pak4 to phosphorylate an established substrate , the proapoptotic protein bad ( gnesutta et  al . , 2001 ) , upon mutation of either pak4 or its phosphorylation site in bad ( ser112 to thr , resulting in bad ) .
coexpression of wt pak4 catalytic domain with bad in hek293 cells yields robust phosphorylation at ser112 ( figure  2 ) . despite having similar catalytic parameters to the wt kinase in  vitro on peptide substrates ( see table 1 ) , pak4 expression
in contrast , pak4 could phosphorylate bad in cells , but wt pak4 phosphorylation of bad was substantially reduced .
these results suggest that an optimal combination of phosphorylation site residue and dfg+1 residue is essential for maximal phosphorylation of at least some kinase substrates in living cells . to understand the structural basis for how the dfg+1 residue controls phosphoacceptor specificity , we determined the x - ray cocrystal structures of wt and f461v mutant pak4 catalytic domains in complex with consensus peptide substrates incorporating both their favored and disfavored phosphorylation site residues ( paktide - s and paktide - t , figure  3 ) .
we also determined an additional x - ray cocrystal structure of wt pak4 with a longer version of the consensus peptide , paktide - s(l ) ( figure  s3 ) .
as previously observed for wt pak4 in the absence of bound peptide ( eswaran et  al .
, 2007 ; ha et  al . , 2012 ) , in each of the structures pak4 is observed in the active state conformation with the activation loop phosphorylated on residue ser474 .
the maximal root - mean - square deviation between the five structures is 1.1   over 290 c- atoms ( table 2 ) .
interestingly , the cocrystallized atp analog was only visible in the electron density for the structure of the wt pak4 complex with paktide - s ( figure  s3 ) .
both structures of wt pak4 in complex with the optimal substrate , paktide - s or paktide - s(l ) , are highly similar , with small differences observed in the conformation of several side chains within the bound peptide and some crystal packing - induced conformational differences c - terminal to the phosphoacceptor residue ( figure  s3 ) .
the bound peptide has a nearly identical backbone conformation , and , importantly , the residues most strongly selected by the kinase ( the p-2 arg and the p0 ser residues [ rennefahrt et  al . , 2007 ; zhu et  al . , 2005 ] ) are observed in very similar orientations . as found in other structures of ser - thr kinases with bound peptide substrates ( brown et  al . , 1999 ;
, 2005 ; madhusudan et  al . , 1994 ; yang et  al . ,
2002 ) , in both structures the ser hydroxyl points toward the atp binding site and is engaged in a network of polar contacts with conserved residues in the kinase catalytic loop ( figures 3a and s3l ) .
both structures therefore show the pak4-peptide complex in a conformation apparently competent for phosphate transfer to occur .
the dfg+1 phe residue in these  wt pak4 structures is positioned within 4 of the ser phosphoacceptor -carbon and oriented perpendicular to the substrate peptide backbone ( figure  3b ) .
this conformation for the dfg+1 phe would preclude the additional methyl group of a thr phosphoacceptor due to steric hindrance .
indeed , in the structure of wt pak4 in complex with paktide - t , the bound peptide is in a nearly identical conformation .
however , to accommodate the thr phosphoacceptor residue , the dfg+1 phe undergoes significant conformational movement : it rotates 75 about its - bond to allow the face of the phe aromatic ring to interact with the thr methyl group ( figure  3c ) .
this conformation for the dfg+1 residue likely interferes with positioning of the -phosphate of atp ( figures s3m  s3o ) .
consistent with the kinetic data , the structure does not suggest impaired paktide - t binding to the wt kinase . rather , decreased phosphorylation of thr substrates may occur due to conformational effects within the catalytic center . taken together , these structures provide a likely explanation for why wt pak4 , and by extension other kinases with a dfg+1 phe residue , is selective for ser at the phosphorylation site . to understand why kinases with a -branched dfg+1 residue favor thr , we examined the cocrystal structures of pak4 in complex with paktide - s and with paktide - t . in the cocrystal structure of pak4 with paktide - t
, the phosphoacceptor thr methyl group stacks snugly against the mutated dfg+1 val residue , positioning the phosphoacceptor hydroxyl in an orientation similar to that observed in the wt pak4-paktide - s complex ( figure  3d ) .
the phosphoacceptor thr appears poised for phosphate transfer , and thus the combination of val at the dfg+1 position and thr at the phosphoacceptor site seems to be highly accommodative to positioning an active - like conformation for the phosphoacceptor hydroxyl . in striking contrast
, the cocrystal structure of pak4 in complex with paktide - s displays an unexpected conformation of the phosphoacceptor ser residue ( figure  3e ) .
although the overall peptide conformation is similar , the phosphoacceptor ser is rotated about its - bond ( 1 ) such that the hydroxyl group is pointed away from the active site .
the pak4 cocrystal structures therefore suggest that val at the dfg+1 residue favors thr over ser because the additional methyl group allows interplay between the dfg+1 and phosphoacceptor residues , correctly positioning them to promote an optimal conformation for phosphate transfer .
our results suggest that discrimination by kinases between ser and thr involves control of proper conformation rather than binding to the active site .
our conclusion that ser and thr peptides bind with equal affinity is partly based on our observation that pak4 and mst4 phosphorylate ser and thr substrates with similar km values .
however , because the km value for an enzyme reaction is not necessarily equivalent to the substrate dissociation constant , substrate binding , conformation , and product release could all theoretically contribute to phosphoacceptor discrimination .
we note , however , that ser and thr peptides appear to bind with equal affinity to the ser - selective kinase pka ( aimes et  al . , 2000 ) .
discrimination between tyr and ser / thr by kinases appears to involve differences in binding affinity as well as conformational control .
x - ray crystal structures of tyr kinases in complex with substrates have revealed favorable nonpolar interactions between the phosphoacceptor tyr and conserved tyr kinase - specific residues within the active site cleft ( favelyukis et  al . , 2001 ; hubbard , 1997 ) .
in addition , tyr kinases and ser - thr kinases differ in the conformation of the so - called p+1 loop , which interacts with the peptide backbone of a bound substrate to control its distance from the active site , thus dictating the size of the phosphoacceptor that can be accommodated ( hubbard , 1997 ; taylor et  al . , 1995 ) .
comparative kinetic analyses have been conducted for ser and tyr peptide phosphorylation by casein kinase 2 ( ck2 ) , a kinase that belongs to the cmgc ser - thr kinase group but that can also phosphorylate on tyr residues , albeit with greatly reduced catalytic efficiency .
these analyses revealed large ( 100-fold or more ) differences in both kcat and km , as well as distinct sequence preferences at residues flanking the phosphoacceptor ( marin et  al .
these results suggest that ser and tyr peptides bind in distinct modes to the active site of ck2 and imply different binding affinities .
in contrast , we found that ser and thr peptides bound to pak4 with an identical backbone conformation and apparently comparable affinities .
the preference of a kinase for ser or thr , as reflected in peptide substrate phosphorylation in  vitro , is likely to be generally important for substrate targeting in  vivo .
for example , we observed that most kinases in yeast prefer ser , and based on the identity of their dfg+1 residues , most human kinases are predicted to prefer ser as well .
these observations suggest that the preponderance of pser among yeast and human phosphoproteomes ( hornbeck et  al . , 2012 ;
sadowski et  al . , 2013 ) is related to intrinsic biochemical properties of eukaryotic kinases .
for some kinases the presence of the preferred phosphoacceptor residue may constitute an essential component of its phosphorylation site motif .
however , in many cases the preferred phosphoacceptor , while not absolutely required for phosphorylation by the kinase , may serve to confer robustness on substrate phosphorylation .
for example , recent work has shown that exchanging thr for ser at sites of phosphorylation by the mtor kinase could modulate sensitivity to activating stimuli or inhibitors of the kinase ( kang et  al . , 2013 ) .
in addition to influencing recognition by kinases , phosphorylation site identity can also influence downstream signaling as both protein phosphatases and phosphoprotein interaction domains can discriminate between pser and pthr ( bremmer et  al .
while we have identified the dfg+1 residue as a major determinant for several kinases , other residues within the catalytic domain undoubtedly contribute to phosphoacceptor preference as well .
individual kinases vary widely with respect to the stringency with which they discriminate between the two residues , and this is likely to be controlled by additional active site residues .
for example , a residue in the glycine - rich loop located proximal to the -phosphate of bound atp was shown to influence the extent to which pka prefers ser ( aimes et  al . , 2000 ) .
in addition , we found that a dfg+1 leu residue correlated with ser preference for most kinases , yet for kinases in the cmgc group it was associated with a lack of phosphoacceptor selectivity .
identification of additional  modifier  residues will facilitate a more complete understanding of structural features controlling kinase specificity .
in addition , for some kinases it is possible that phosphorylation site specificity is entirely determined by other residues .
nonetheless , our current study suggests that , by inspection , one may determine whether a kinase is selective for ser or thr . to wit , a -branched residue at the dfg+1 position appears to invariably dictate a preference for thr , but phe dictates preference for ser as the phosphoacceptor residue .
our study therefore provides a much improved understanding of how protein kinases discriminate between phosphorylation site residues .
pak4 , mst4 , and pka ( wt and point mutants ) were expressed with n - terminal hexahistidine tags in e.  coli and purified by immobilized metal affinity chromatography .
pak4 and mst4 were further subjected to anion exchange , followed by size exclusion chromatography or size exclusion chromatography alone , respectively .
gst - snf1 ( wt and mutant ) was expressed in e.  coli , purified by one - step glutathione affinity chromatography , and then activated by phosphorylation with recombinant elm1 kinase , as described ( lee et  al . , 2012 ) .
descriptions of expression vectors used and detailed purification procedures are provided in supplemental experimental procedures .
kinases were incubated with peptides and [ -p]atp at 30c for varying times , and aliquots were spotted on to p81 filter discs .
filters were quenched in 75  mm phosphoric acid and then washed three times for 4  min in 75  mm phosphoric acid , washed once briefly in acetone , air - dried , and analyzed by liquid scintillation counting .
the amount of product formed was calculated from a standard curve made using [ -p]atp at the same specific activity .
initial rates were determined by fitting the data to a line with excel , and catalytic constants were calculated by fitting the initial velocity data to the michaelis - menten equation using kaleidagraph software .
detailed procedures , including buffer compositions for each kinase reaction , are provided in supplemental experimental procedures .
hek293 cells were cultured at 37c and 5% co2 in dulbecco s modified eagle s medium containing 4.5 g / l d - glucose and l - glutamine ( invitrogen ) , 10% fetal bovine serum ( fbs ) , and 1% penicillin / streptomycin .
cells in 6-well plates were cotransfected with 0.4  g pegfp - pak4 catalytic domain and 3.6  g pebg - bad using lipofectamine 2000 ( invitrogen ) . after 24  hr
, cells were exchanged into reduced serum medium ( 0.1% fbs ) for 16  hr and treated with 250  nm wortmannin for 30  min prior to lysis .
cell lysates were prepared by washing briefly with pbs and extracting into lysis buffer ( 20  mm tris - hcl [ ph 7.5 ] , 150  mm nacl , 1  mm edta , 1  mm egta , 1% triton x-100 , 2.5  mm sodium pyrophosphate , 1  mm -glycerophosphate , 1  mm na3vo4 , 1  mm dtt , 1  mm pmsf , 10  g / ml leupeptin , 2  g / ml pepstatin , and 10  g / ml aprotinin ) for 10  min at 4c with rotation and centrifuging ( 10  min at 13,500   g ) .
gst - tagged bad was purified from the supernatant with 20  l glutathione sepharose 4b suspension ( ge healthcare ) .
samples were subjected to sds - page followed by immunoblotting with -bad pser112 antibody ( cell signaling technology # 9291 ) , -gst antibody ( cell signaling technology # 2624 ) and -gfp antibody ( clontech # 632381 ) , and detection by enhanced chemiluminescence .
crystals of wt pak4 and pak4 catalytic domain were obtained with both paktide - t ( ggrrrrrtwyfgggk ) and paktide - s ( rrrrswy ) using the vapor diffusion hanging drop method .
for wt pak4 with peptides , we grew wt pak4 crystals against 0.1  m tris - hcl ( ph 7.5 ) and 1.52.0  m naoac at room temperature and soaked with peptides . for pak4 cocrystals with peptide
, we grew against conditions of 0.1  m tris - hcl ( ph 7.5 ) and 1.52.0  m naoac at room temperature . for cocrystals of pak4 in complex with paktide - s(l ) ( rrrrrswyfdg ) , we grew against 1.7  m ammonium sulfate , 15% peg400 , and 0.1  m tris - hcl ( ph 8.0 ) .
24-id - e , national synchrotron light source ( nsls ) beamline x25 , or the swiss light source beamline sls - x10 .
following structure solution , good electron density was observed for each of the kinase domains and their bound peptides .
 summaryeukaryotic protein kinases are generally classified as being either tyrosine or serine - threonine specific . 
 though not evident from inspection of their primary sequences , many serine - threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue . here 
 we show that a residue located in the kinase activation segment , which we term the  dfg+1  residue , acts as a major determinant for serine - threonine phosphorylation site specificity . 
 mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases , including the serine - specific kinase pak4 and the threonine - specific kinase mst4 . 
 kinetic analysis of peptide substrate phosphorylation and crystal structures of pak4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate . 
 rather , favored kinase - phosphoacceptor combinations likely promote a conformation optimal for catalysis . 
 understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence . 
Introduction
Results
Discussion
Experimental Procedures