cellular adaptation requires biochemical processes including post - translational mechanisms to modify existing proteins . catalyzed by opposing kinases and phosphatases , reversible phosphorylation of serine , threonine , and tyrosine residues is now appreciated as a fundamental regulatory mechanism with the majority of phosphorylation ( > 99% ) occurring on serine and threonine residues [ 1 , 2 ] . due to their untapped therapeutic potential ,
protein phosphatases have been identified as promising targets for xenobiotic manipulation through rational drug design ( reviewed in [ 36 ] ) . in particular ,
the ubiquitously expressed protein phosphatase 2a ( pp2a ) has been proposed as a target for the treatment of a number of pathologies ranging from neurodegenerative diseases such as alzheimer disease to a variety of neoplasias [ 79 ] .      
compared to other members of the phosphoprotein phosphatase ( ppp ) superfamily of serine / threonine phosphatases , a detailed understanding of the mechanism by which pp2a recognizes substrates and mediates site - specific dephosphorylation remains to be developed . sequence and structural homology of the catalytic subunits of ppp family members
has revealed a conserved catalytic mechanism in which a divalent metal cation activates a water molecule to hydrolyze phospho - serine / threonine without the formation of a phosphoenzyme intermediate ( reviewed in [ 1012 ] ) . despite a shared catalytic mechanism , substrate specificity within the ppp family
for example , the ppp calcineurin ( also known as protein phosphatase 2b ) has been shown to interact with two consensus sequences , pxixit and lxvp , found on nonsubstrate - interacting proteins and target substrates ( reviewed in [ 11 , 13 ] ) . for protein phosphatase-1 ( pp1 ) , substrate specificity is conferred by incorporation of pp1-interacting proteins via a conserved docking motif with a general consensus sequence of rvxf ( reviewed in [ 11 , 12 ] ) . at present , consensus sequences in pp2a substrates have not been identified .
this review will focus on our emerging understanding of pp2a substrate specificity , which appears to involve additive effects of multiple discrete interactions .          
pp2a is a highly conserved serine / threonine phosphatase which , depending on the tissue of origin and cell type , may account for up to 1% of cellular protein and the majority of serine / threonine phosphatase activity .
the physiological functions of pp2a have been implicated in all facets of cellular existence ( reviewed in ) .
further , pp2a functions as a critical tumor suppressor whose interruption leads to proliferative diseases .
the heterotrimeric holoenzyme is composed of a catalytic subunit ( c ) a scaffold subunit ( a ) and one member of four families of regulatory subunits ( b ) ( figure 1 ) .
the diversity of pp2a heterotrimers is achieved through expression of two c subunits , two a subunits and approximately fifteen b subunits in vertebrates .
the b subunits are derived from four diverse gene families ( b , b , b , and b ) that have little sequence similarity between families but maintain high sequence similarity within families .
the b family ( b55 , pr55 , ppp2r2 ) of regulatory subunits consists of four genes (  ,  ,  ,  ) , the b - family ( b56 , pr61 , ppp2r5 ) is comprised of five isoforms (  ,  ,  ,  ,  ) , the b family ( pr72 , ppp2r3 ) includes three isoforms ( /pr72/130 , /pr59 , /pr48 ) , and the b family ( pr93/pr110 ) is made up of three proteins ( sg2na , striatin , and mmob1 ) .
there is some controversy as to whether the b family members , most notably sg2na , are bona fide pp2a regulatory subunits that always associate with the ac dimer or whether they are merely regulated by association with the pp2a dimer .
given the large number of pp2a subunits , it is thought that each cell expresses a dozen or more distinct holoenzyme complexes which act on a diverse array of substrates .
pp2a holoenzyme diversity has been the subject of several excellent papers [ 14 , 15 , 18 ] . like pp1 , regulatory subunit incorporation is thought to dictate the substrate specificity of the pp2a complex , however , only recently have molecular studies begun to develop insight into the mechanism by which the regulatory subunit acts .
the results from recent studies suggests a multitiered mechanism wherein pp2a substrate specificity arises from ( 1 ) subcellular localization of pp2a defined by the b subunit , ( 2 ) selective holoenzyme assembly by posttranslational modification , ( 3 ) interaction with specific endogenous inhibitors , ( 4 ) interactions between the b subunit and phosphosubstrates at sites distant from the active site , and ( 5 ) b - subunit residues which infiltrate the catalytic cleft of the c subunit .
this paper will provide a summary of these studies and how the understanding of the determinants of pp2a substrate specificity has advanced .
the heterotrimeric holoenzyme is targeted to discrete subcellular locales dictated in part by which b - regulatory subunit is incorporated .
the localization imparted by the b - regulatory subunit dictates the spatial sphere of influence of the holoenzyme complex for potential substrates .
this mechanism of targeting pp2a activity is highlighted by extensive studies of the b family of regulatory subunits .
for instance , the b family regulatory subunits target the holoenzyme to different cellular compartments in the brain . specifically , b and b are primarily cytosolic where as the b-regulatory subunit associates with a detergent - resistant protein fraction consistent with an interaction at the cytoskeleton
.    similar diversity has been observed in the b family of regulatory subunits . a c - terminal nuclear export signal common to b , b , and b which , when these regulatory subunits are incorporated into the pp2a holoenzyme , results in cytoplasmic localization of the heterotrimer . b and b isoforms lack a similar sequence and are found primarily in the nucleus . in cardiomyocytes , b interacts with the protein ankyrin - b through its c - terminus which leads to localization at the cardiac m - line .
b on the other hand has been shown to target the holoenzyme complex to subnuclear structures in cardiomyocytes where pp2a / b may regulate gene expression .
loss of proper subcellular targeting of pp2a has been implicated in the biogenesis and aggressive phenotype of neoplastic growths .
specifically , a truncated form of b ( b ) , has been isolated from a melanoma cell line wherein the pp2a/b complex is targeted to the trans - golgi network , blunts p53 responsiveness , contributes to genetic instability and increases metastatic motility [ 2427 ] .    
this was first observed in a member of the b family , b. two neuron - specific isoforms of b , b1 , and b2 , are generated by the use of an alternative 5 exon which results in the production of a divergent n - terminal extension on b2 [ 28 , 29 ] .
the n - terminal extension of b2 directs the holoenzyme complex to the outer mitochondrial membrane ( omm ) by targeting the mitochondrial translocase complex and forming an abortive complex resistant to import into the mitochondrial matrix [ 28 , 30 ] .
the omm - directed pp2a / b2 complex promotes fragmentation of the mitochondria reticulum and increases cell susceptibility to proapoptotic insults through an unknown mechanism .
alternative splicing of b - regulatory subunits directing subcellular localization of the pp2a holoenzyme has also been observed in both the b and b families .
two b isoforms differ by the inclusion or exclusion an n - terminal nuclear localization signal leading to isoform - specific nuclear or cytoplasmic localization .
the murine - specific b family member , b , is also subject to alternative splicing leading to isoform - specific nuclear or cytoplasmic localization through an as yet unidentified mechanism .
for example , the interaction between pp2a / b and shugoshin during meiosis is crucial for spatial and temporal regulation of sister chromatid disjunction . during meiosis , the cohesin complexes , which link the arms of bivalent chromosomes and the centromeres of sister chromatids ,
must be released in a stepwise fashion by the protease separase ; the cohesin complex is firstly hydrolyzed along the arms of the bivalent chromosomes for completion of anaphase i and secondly at the centromeres of sister chromatids for completion of anaphase ii . during anaphase
i , the centromeric cohesin complex is protected from separase - dependent proteolysis by shugoshin [ 34 , 35 ] .
shugoshin recruits pp2a / b to the centromere which likely results in dephosphorylation of the cohesin complex leading to protection of the cohesin complex from separase - dependent proteolysis [ 36 , 37 ] . through cocrystallization ,
xu and colleagues , have revealed that dimeric human shugoshin 1 interacts with pp2a / b through a coiled - coil region across a broad composite surface of the c and b subunits .
incorporation of specific regulatory subunits is influenced by reversible posttranslational modification of the c subunit .
many groups have shown that the c - terminus of the c subunit is modified through phosphorylation and methylation on y307 and l309 , respectively [ 3946 ] .
phosphorylation of y307 is catalyzed by src kinase and is likely opposed by pp2a - catalyzed autodephosphorylation of this phosphotyrosine .
phosphorylation of y307 selectively inhibits recruitment of the b family and some b family members to the dimeric ac complex whereas b recruitment is not effected .   
methylation of the c subunit at the c - terminal l309 is catalyzed by the protein phosphatase methyltransferase ( ppm1 ) and is opposed by the phosphatase methylesterase ( pme-1 ) [ 4751 ] .
reversible methylation of pp2a is absolutely critical as knocking - out pme-1 in mice changes the phosphoproteome and results in early perinatal lethality .
further , methylation of the c subunit is a dynamic process which plays a role in cellular response to acute stimuli .
the recruitment of the b subunit to the ac dimer has been postulated to require methylation of the c subunit for some of the b - subunit families .
however , conflicting results have been reported that may reflect differences in experimental design and will be discussed further .
studies wherein pp2a / b is isolated from intact cells have revealed that methylation of the c subunit at l309 is required for incorporation of the b family of regulatory subunits into the holoenzyme complex [ 39 , 40 , 42 , 44 , 54 , 55 ] .
conversely , in vitro assembly of the pp2a / b holoenzyme complex does not require methylation of the c subunit for incorporation of the b family of regulatory subunits [ 56 , 57 ] .
similarly controversial , the requirement for c subunit l309 methylation was observed to be study - specific in in vitro pp2a / b timer formation [ 58 , 59 ] .
methylation was dispensable for isolation of the pp2a / b holoenzyme complex from intact cells .
the role of methylation of the c subunit in recruitment of b and b families of regulatory subunits is less controversial with methylation of the c subunit being dispensable [ 44 , 54 ] . for more information ,
posttranslational modifications which influence formation of the pp2a holoenzyme complex also occur on the b subunit .
pp2a / b negatively regulates the erk map kinase signal transduction pathway . through formation of a ternary complex of the early response gene product iex-1 , b1 , and erk ,
erk mediates its own disinhibition by phosphorylation of b1 on s327 leading to b1 disassociation from the pp2a holoenzyme .
since s327 is conserved among b - subunit family members , it is likely that other b subunits are regulated similarly .
additionally , b is likely phosphorylated on s167 to disrupt the b subunit from the ac dimer in early mitotic stages .
however , pp2a / b activity is necessary to resolve the mitotic spindles and conclude mitosis ; therefore , autodephosphorylation may occur on b to allow efficient pp2a / b heterotrimer formation and cell cycle progression .
thus , phosphorylation of the b - regulatory subunits also influences holoenzyme assembly and , therefore , substrate specificity .
phosphorylation of the b subunit of heterotrimeric pp2a also potentiates the catalytic activity of the holoenzyme complex . in response to activation of d1 dopamine receptors on striatal neurons ,
camp - dependent protein kinase a ( pka ) phosphorylates    b at s566 increasing activity of the pp2a/ b holoenzyme towards dopamine- and camp - regulated neuronal phosphoprotein ( darpp-32 ) [ 64 , 65 ] .
dephosphorylation of t75 on darpp-32 by pp2a / b disinhibits pka - mediated phosphorylation of darpp-32 at t34 which converts darpp-32 into a potent pp1 inhibitor leading to changes in neuronal signaling .
this circuit acts to attenuate phospho - t75 inhibition of t34 phosphorylation of darpp-32 . this circuit has been shown to be differentially regulated by psychomotor stimulants and antipsychotics acting on different striatal neuron subpopulations .   
while assembly of the many trimeric pp2a holoenzymes directs cellular localization and substrate specificity , further regulation is afforded through binding of specific protein inhibitors of pp2a .
one such inhibitor set ( i2/taf-1 ) is upregulated during the progression of chronic myelogenous leukemia through bcr / abl activity and results in decreased pp2a activity .
an additional pp2a inhibitor is the protein cip2a ( cancerous inhibitor of pp2a ) , overexpression of which is associated with several human malignancies .
cip2a associates with c - myc to protect its phosphorylated s62 from pp2a - directed activity stabilizing the c - myc protein and allowing it to promote oncogenesis .   
recently an interplay between the drosophila serine / threonine kinase greatwall ( gwl ) and pp2a / b was observed during mitotic entry in two separate studies [ 69 , 70 ] .
pp2a / b activity prevents mitotic entry by maintaining cdc25 in a dephosphorylated and inactive state .
gwl reverses this inhibition through the phosphorylation of s67 of both -endosulfine ( ensa ) and cyclic adenosine monophosphate-(camp- ) regulated phosphoprotein-19 ( arpp-19 ) .
phosphorylation converts ensa and arpp-19 into very specific inhibitors of pp2a / b activity and produces activation of cdc25 leading to cell cycle progression .
similar cell cycle regulatory activity has been observed with the mammalian ortholog of gwl , mastl ; however , the mastl - pp2a interaction has yet to be characterized .
once targeted to specific subcellular locales , the pp2a holoenzyme must recruit and dephosphorylate target substrates .
recent structural studies have begun to suggest the mechanism by which the regulatory subunit of pp2a mediates initial binding to target substrates .
other -propeller proteins have been shown to bind ligands in the central depression on the top surface of the toroid .
crystallization of pp2a / b revealed a cluster of acidic residues in this depression that is available to recruit potential substrates containing a basic motif . in this same study ,
the acidic central depression of b was experimentally confirmed to bind the microtubule - associated protein tau , an established pp2a / b substrate .
several conserved aspartate and glutamate residues in b engage in weak , electrostatic interactions across a large basic portion of tau and support dephosphorylation of tau at multiple sites through cycles of binding and unbinding .    structurally divergent b family members may recruit substrates in a similar fashion as the b family .
the crystal structure of pp2a / b shows that the b subunit contains 18 stacked -helices which adopt 8 huntingtin elongation a subunit tor- ( heat- ) like repeats [ 58 , 59 ] .
a portion of these heat - like repeats interact with the a subunit of the holoenzyme to mediate regulatory subunit incorporation into the holoenzyme complex . like the b subunit
, an acidic patch is exposed in the b family of regulatory subunits and may mediate protein - protein interactions and substrate recruitment .
structural studies of pp2a have revealed a conserved loop in the b family of regulatory subunits which infiltrates the catalytic core of the holoenzyme [ 58 , 59 ] ( figure 2(a ) ) . at the tip of this loop
is a conserved glutamate residue , e153 ( b numbering ) , which contacts through its carbonyl oxygen the catalytic subunit and through its carboxyl group a cocrystallized microcystin molecule in the active site .
mutational analysis revealed that e153 is an absolute requirement for efficient dephosphorylation of tyrosine hydroxylase ( th ) , a known pp2a / b substrate , as well as other as yet unidentified cellular substrates of this pp2a holoenzyme .
further , it was determined that e153 of b interacts with r37 and r38 of th to mediate dephosphorylation of both s31 and s40 on th .
positively charged residues in the vicinity of target phospho - serine / threonine residues could represent a consensus sequence for b - subunit - mediated dephosphorylation .
further , the infiltrating loop is likely a conformationally dynamic structure which is not sterically hindered by surrounding portions of the a , c , or b subunits .
since r37/38 are important for dephosphorylation of both upstream ( s40 ) and downstream ( s31 ) sites , it appears that the orientation of phosphopeptides relative to the catalytic cleft is not constrained by additional interactions .
collectively , the above observations support a model in which the sites of interaction between the substrate and the b regulatory subunit that are distant and near the active site together control substrate specificity .
first , the interaction occurring at sites distant from the active site increases the local substrate concentration . following this initial substrate recruitment ,
although divergent in its sequence , an analogous structure from the -propeller fold of the b family of regulatory subunits extends to the catalytic core of the holoenzyme   ( figure 2(b ) ) .
this loop places conserved residues of the b - family subunits very near the holoenzyme active site . unpublished observations generated in our lab suggests that of these loop residues k87 of b2 may play a similar role as e153 of b in site - specific dephosphorylation of target substrates ; however , further characterization of this substrate specificity loop is required .
pp2a is a ubiquitous protein phosphatase responsible for the dephosphorylation of many different intracellular targets . the diverse repertoire of potential substrates for pp2a is imparted by the incorporation of one of fifteen unique b - regulatory subunits .
recent studies have increased our understanding of the mechanisms by which the b subunit imparts specificity to the holoenzyme complex . through selective incorporation of the b
- regulatory subunit , the holoenzyme complex is recruited to discrete subcellular locales which define the sphere of influence for the phosphatase .
secondly , interactions between endogenous inhibitors and specific pp2a heterotrimers further restrict phosphatase activity . as shown for the b family , regulatory subunits mediate low - affinity interactions with substrates to increase the local concentration of substrates . through a flexible substrate selectivity loop which contacts the catalytic subunit
, interactions between the regulatory subunit and phosphosubstrate may mediate multiple nearby dephosphorylation events . with the current structural information available for
the pp2a complexes , future high - resolution studies will further define the molecular mechanism of pp2a substrate specificity . as general ,
inhibitors of pp2a are either clinically irrelevant or toxic , as in the case of the small molecule inhibitor microcystin , novel methods to increase the specificity of pp2a inhibition or activation must be developed .
a clearer understanding of the pp2a substrate specificity mechanisms will serve as the foundation for rational drug design of selective inhibitors and activators of specific pp2a holoenzyme complexes .
 protein phosphatase 2a- ( pp2a- ) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function . 
 pp2a is predominantly found as a heterotrimeric complex of a catalytic subunit ( c ) , a scaffolding subunit ( a ) , and one member of 4 families of regulatory subunits ( b ) . 
 substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to , selective incorporation of the b subunit , interactions with endogenous inhibitory proteins , and specific intermolecular interactions between pp2a and target substrates . here 
 , we discuss recent studies that have advanced our understanding of the molecular determinants for pp2a substrate specificity . 
1. Introduction
2. Mechanisms of Substrate Specificity
3. Conclusion