Báo cáo khoa học: Phosphorylation mechanism and structure of serine-arginine protein kinases pot

SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10–12 serines in the N-terminal region of the RS domain RS1, using a mechanism that incorporates sequential, C-te

Phosphorylation mechanism and structure of

serine-arginine protein kinases

Gourisankar Ghosh1and Joseph A Adams2

1 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

2 Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA

Introduction

The complexity of the human proteome is regulated

through the alternative splicing of large precursor

mRNAs [1] Although this process plays a significant

role in normal cellular development, changes or defects in alternative splicing have also been linked to human disease [1–3] Splicing reactions occur at the

Keywords

mechanism; protein kinase; splicing;

SR protein; structure

Correspondence

J A Adams, Department of Pharmacology,

University of California, San Diego, La Jolla,

CA 92093-0636, USA

Fax: +1 858 822 3361

Tel: +1 858 822 3360

E-mail: j2adams@ucsd.edu

(Received 7 July 2010, revised 9 November

2010, accepted 10 December 2010)

doi:10.1111/j.1742-4658.2010.07992.x

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain) SR proteins are phosphory-lated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events Although SR proteins are polyphosphorylated in vivo, the mecha-nism underlying this complex reaction has only been recently elucidated Human alternative splicing factor [serine⁄ arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phos-phorylated by SRPK1, a post-translational modification that controls cyto-plasmic–nuclear localization SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10–12 serines in the N-terminal region

of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound

to a docking groove in the large lobe of the kinase domain Upon subse-quent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a b-strand in RNA recognition motif 2 unfolds and occupies the docking groove These studies indicate that efficient regiospec-ific phosphorylation of SRSF1 is the result of a contoured binding cavity

in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism

Abbreviations

CLK, cdc2-like kinase; kdSRPK1, kinase-dead form of human alternative splicing factor; LysC, lysyl endoproteinase; MAPK, mitogen-activated protein kinase; RRM, RNA recognition motif; RS domain, domain rich in Arg-Ser repeats; RS1, N-terminal region of human alternative splicing factor domain rich in Arg-Ser repeats; RS2, C-terminal region of human alternative splicing factor domain rich in Arg-Ser repeats; SRPK, SR-specific protein kinase; SRSF, serine ⁄ arginine-rich splicing factor; SRSF1, human alternative splicing factor (serine ⁄ arginine-rich splicing factor 1).

spliceosome, a macromolecular complex composed of

five small nuclear ribonucleoproteins (U1, U2, U4, U5,

and U6) and over 100 auxiliary proteins [4] Among

these many proteins, one family of splicing factors,

known as SR proteins, is essential for controlling

numerous aspects of mRNA splicing as well as other

RNA processing events SR proteins interact with

splicing components (U1-70K and U2AF35) early

during spliceosome development, and help to establish

the 5¢ and 3¢ splice sites [5,6] Later, they recruit the

U4⁄ U6ÆU5 tri-small nuclear ribonucleoprotein [7] and

also enhance the second catalytic step in splicing [8]

Splicing is tightly coupled to transcription, and SR

proteins have been shown to play a role by binding

the C-terminal domain of RNA polymerase II and

reg-ulating CDK9 [9] SR proteins serve roles in many

postsplicing events, including mRNA export [10,11],

translation regulation [12,13], and genomic stability

[14,15] More than a decade ago, it was discovered that

two protein kinase families [SR-specific protein kinases

(SRPKs) and cdc2-like kinases (CLKs)] phosphorylate

SR proteins, altering their cellular distribution and

activities [16–18] In the last several years, great strides

have been made in understanding how the SRPKs

rec-ognize and phosphorylate the SR proteins In this

review, we will highlight how a highly dynamic and

distinct interplay between kinase and substrate is

nec-essary for modification of the SR protein human

alter-native splicing factor [serine⁄ arginine-rich splicing

factor 1 (SRSF1)], a prototypical SR protein involved

both in constitutive and in alternative splicing [19]

These studies have shown that SRPK1 has an

interest-ing feedinterest-ing mechanism, whereby multiple contacts in

the SR protein are utilized to catalyze a lengthy polyphosphorylation reaction

Structural features of SR proteins

SR proteins derive their name from a lengthy (50–300-residue) C-terminal tail rich in Arg-Ser repeats known

as the RS domain (Fig 1) SRSF1 (also known as ASF⁄ SF2) represents a typical arrangement for an RS domain, where Arg-Ser repeats are bracketed by smal-ler repeats and some isolated Arg-Ser pairs In addi-tion to RS domains, SR proteins also contain one or two N-terminal RNA recognition motifs (RRMs) that modulate SR protein interactions in the spliceosome

by binding short mRNA sequences (splicing enhancers) [20,21] Numerous screening procedures have revealed that the observed determinants are somewhat nonspe-cific, raising the possibility that members of the SR protein family serve redundant functions in mRNA splicing [22] Although, in support of this idea, all SR proteins can complement splicing-deficient S100 cyto-plasmic extracts of HeLa cells [20], there are other studies showing that certain SR proteins play tissue-specific roles at various developmental stages [23,24], arguing for a specialized role for some SR proteins Although there is no X-ray structure for an SR protein

in either a phosphorylated or unphosphorylated state,

a recent NMR structure shows that one of the RRMs

of SRSF1 (RRM2) adopts a typical RNA-binding fold [25] Sequence analyses suggest that SR proteins may have properties consistent with intrinsically disordered proteins, owing to an RS domain that is expected to

be largely unstructured [26] On the other hand, all

RS domain

RRM domain

P RS P YG RSRSRSRSRSRSRSRS N SRSRS Y PR RS RG S PRY S PRH SRSRS RT-C′

SRPK phosphorylation

nuclear entry/speckle formation

CLK phosphorylation nuclear dispersion

Fig 1 SR protein domain structure All traditional SR proteins have one or two N-terminal RRMs and one C-terminal RS domain The amino acid sequence for the

RS domain of the prototype SR protein SRSF1 is displayed Peptide mapping and cellular analyses indicate that the phosphor-ylation of two segments in this RS domain (RS1 and RS2) by SRPKs and CLKs control the subcellular distribution.

atom calculations of an eight dipeptide repeat

[(Arg-Ser)8] suggest that the unphosphorylated sequence

adopts a helical form, with the arginines pointing out

into solution for charge minimization, and a compact,

‘claw-like’ structure upon phosphorylation [27]

Appre-ciable helical content has not been detected in CD

experiments for SRSF1 or its RS domain in either the

phosphorylated or unphosphorylated forms [28],

sug-gesting that if the Arg-Ser repeats possess helical

struc-ture, it may not be highly stable in solution Recent

studies have shown that the phosphorylated RS

domain is protected from dephosphorylation by the

neighboring RRMs, suggesting that the RS domain

may not be disordered and could pack onto other

domains in the SR protein [29] Nonetheless, although

the RRMs adopt a classic RNA-binding fold, it is still

not fully clear how the RS domain folds by itself or in

the context of the SR protein, or how phosphorylation

modifies the SR protein conformation

SR proteins are phosphorylated by two

protein kinase families

Early studies showed that SR proteins undergo

multi-ple rounds of phosphorylation and dephosphorylation

en route to spliceosome assembly [30–32]

Phosphoryla-tion was shown to occur in the RS domain and alter

how the SR protein functions in the spliceosome For

example, the SR proteins SRSF1 and SRSF2 (also

known as SC35) interact with the 70-kDa subunit of

U1 (U1-70K) and the 35-kDa subunit of U2AF

(U2AF35) in a phosphorylation-dependent manner

[5,6], establishing the appropriate splice sites More

than a decade ago, it was discovered that the SRPK

and CLK families of protein kinases can

polyphospho-rylate RS domains and alter SR protein cellular

distri-bution and splicing function [16,17,33] However, the

role of RS domain phosphorylation in alternative

splic-ing is not well understood Although some studies have

suggested that the RRMs are the principal driving

ele-ments for alternative splicing of some precursor

mRNAs [34], other studies have shown that the

phos-phoryl content of the RS domain is important For

example, phosphorylation of SRSF1 controls the

alter-native splicing of the caspase-9 and Bcl-x genes and

induction of a proapoptotic phenotype [35] Although

further investigations are needed to provide a more

forceful link between RS domain modification and

splicing, it has become abundantly clear that

phosphor-ylation is directly linked to the nuclear entry of SR

pro-teins It has been shown that phosphorylation of the

RS domain leads to enhanced interactions with the

nuclear import receptor, transportin SR, and entry of

the SR proteins into the nucleus, where they largely reside in speckles [36–38] Whereas SRPKs play a direct role in nuclear import, the CLK family controls the nuclear distribution of SRSF1 and other SR proteins Thus, through interactions with two families of protein kinases, the cellular location and, presumably, splicing function of SR proteins can be precisely controlled Kinetic studies on SRPK1 and SRSF1, together with crystal structures of SRPK1 bound to peptide and pro-tein substrates and the recent structures of CLK1 and CLK3, suggest an elegant mechanism of recognition and phosphorylation by these two kinases, which regu-late the biological function of SR proteins in the cell

SRPK1 structure Most of our knowledge regarding SRPKs comes from studies on SRPK1 and the yeast analog, Sky1p SRPKs contain a well-conserved kinase domain that is bifurcated by a large, nonconserved insert domain (approximately 250 amino acids) The insert domain in SRPKs regulates subcellular localization, as its deletion changes the distribution pattern of the kinase from nuclear–cytoplasmic to exclusively nuclear [39] In addition to this important regulatory domain, SRPKs contain N-terminal and⁄ or C-terminal extensions, which are not conserved Deletion of the insert and N-terminal extension does not inactivate the catalytic activity of SRPK1, suggesting that these elements play auxiliary roles [40] The X-ray structure for SRPK1 lacking its N-terminus and most of the insert domain reveals the signature bilobal fold found in all eukary-otic protein kinases (Fig 2A) The small lobe is com-posed mostly of b-strands, and binds the nucleotide (ADP in the SRPK1 structure) The larger lobe is com-posed mostly of a-helices, and provides residues important for substrate binding A short segment of the insert domain connecting the two major kinase lobes is present, and adopts short helical conforma-tions Like other members of the CMGC group of protein kinases, SRPK1 contains a small insert within the kinase domain known as the mitogen-activated protein kinase (MAPK) insert, which connects heli-ces aG and aH (Fig 2A) Although the X-ray struc-ture of SRPK1 was solved with a short substrate peptide, this peptide binds unexpectedly outside the active site in a groove generated by the MAPK insert and a loop connecting helices aF and aG (Fig 2A) Later, we will discuss how this docking groove binds SRSF1 and feeds the RS domain into the active site for sequential phosphorylation

X-ray structures of the kinase domains of CLK1 and CLK3 have been reported recently [41], and are

worth noting here, given their overlapping substrate

specificities with the SRPKs Although the CLK family

of protein kinases is capable of widespread RS domain

phosphorylation, their structures are distinct from

those of the SRPKs in several ways Most significantly,

the CLK enzymes lack a large insert domain dividing

the kinase core and, unlike the SRPKs, are

auto-phosphorylated on both serine and tyrosine [42] The

CLK kinases have large N-termini, as do the SRPKs,

but, unlike in the SRPKs, these extensions are rich in

isolated Arg-Ser dipeptides Although the CLK family

also belongs to the CMGC group of kinases, changes

in the sequence of the MAPK insert and positions of

helices aG and aH result in the loss of the deep

sub-strate docking groove observed in SRPK1 In addition

to the MAPK insert, CLK1 and CLK3 contain

another small insert between stands b6 and b9 in the

kinase core that interacts with a hydrophobic pocket

near the hinge region connecting the kinase lobes

Maintenance of the constitutively

active conformation

Whereas many protein kinases are highly regulated

through diverse mechanisms, SRPK family members

are constitutively active and require no

post-transla-tional modifications or addipost-transla-tional protein subunits for

optimal activity Several key structural elements are

essential in the maintenance of this highly active form

of SRPK1 In some protein kinases, the activation

loop plays a regulatory role by controlling access to

the active site, and only adopts an open configuration

upon phosphorylation by other protein kinases [43,44]

The activation loop of SRPK1 is comparatively short

and, lacking a reversible phosphorylation site, adopts a

stable conformation that allows ready access of sub-strates to the active site (Fig 2B) Extensive biochemi-cal analyses have shown that the activation loop in SRPK1 is highly malleable [45] Molecular dynamics simulations have shown that alternative residues can mediate contacts that are lost upon mutation of some residues in the activation segment and maintain the structural integrity of the activation segment Thus, SRPK1 is resilient to inactivation, and exhibits robust phosphorylation activity The extensive phosphoryla-tion that SRPK1 must execute for each SR protein is

a likely explanation for the evolution of such robust activity In addition to activation loops, all protein kinases possess a catalytic loop with a conserved aspartic acid that forms a hydrogen bond with the hydroxyl serine⁄ tyrosine of the substrate In the case

of SRPK1, the catalytic loop aspartate is ideally poised

to abstract the hydroxyl hydrogen from the substrate serine, a necessary step for protein phosphorylation [46] Several short-range interactions within the small lobe and between the large and small lobes around the active site participate in maintaining the catalytically active conformation Two conserved interactions in all active protein kinases are also present in SRPK1: an ion pair between an invariant glutamic acid in helix

aC and an invariant lysine in strand b3 in the small lobe, and a hydrogen bond between the activation loop and helix aC (Fig 2B)

Regiospecific phosphorylation of the

RS domain of SRSF1 Although both the SRPK and CLK families catalyze multisite phosphorylation of SR proteins, the struc-tural data suggest that differences in critical regions

Fig 2 Structural features of SRPK1 (A) Ribbon diagram of SRPK1 in complex with ADP and a short peptide substrate (RRRERSPTR) The peptide binds near the MAPK insert SRPK1 lacks most of its N-ter-minus (1–41) and insert domain (256–473) (B) Several conserved structural elements and contacts in SRPK1.

such as the MAPK insert and N-terminus may impart

distinct regiospecificities To address this issue, the

mechanism of phosphorylation of SRSF1 by both

enzymes was investigated with MS methods As shown

in Fig 1, the RS domain of SRSF1 contains many

serines throughout, and it is not clear whether these

two kinases show preferences for specific residues The

mapping of phosphorylation sites in the RS domain is

a vexing problem, owing to the redundancy of the

Arg-Ser repeats and difficulties in separating⁄

identify-ing the closely related polybasic fragments in

tradi-tional mapping studies This problem has been

circumvented by using a modified form of SRSF1 that

contains four Argfi Lys substitutions in the RS

domain Upon phosphorylation and cleavage with lysyl

endoproteinase (LysC), five fragments encompassing

the complete RS domain of SRSF1 could be identified

by MALDI-TOF MS [47] These studies detected

about eight phosphoserines in the N-terminal portion

of the RS domain To further define the

phosphoryla-tion segment in SRSF1, a wide series of truncaphosphoryla-tion

derivatives were made, and their phosphoryl contents

were assessed by MS [48] These studies showed

defini-tively that SRPK1 is a regiospecific protein kinase,

preferring to phosphorylate up to 12 serines in the

N-terminal region of the RS domain of SRSF1 (RS1)

(Fig 1) Single turnover kinetic studies have shown

that RS1 is phosphorylated very efficiently within

1–2 min In comparison, CLK1 does not show this

regiospecificity, and instead can phosphorylate all 20

serines in the RS domain of SRSF1 [47] Furthermore,

CLK1 appears to be able to completely phosphorylate

the RS domain of SRSF1 even if RS1 is

prephosph-orylated by SRPK1 This sequential phosphorylation

of RS1 (by SRPK1) and the C-terminal region of the

RS domain of SRSF1 (RS2) (by CLK1) segments is

biologically relevant, as it has been demonstrated that

SRSF1 lacking RS2 translocates to the nucleus but is

neither additionally phosphorylated nor dispersed in

the nucleus by CLK1 [40] These studies provide a

model in which SRPK1 phosphorylates RS1, leading

to translocation of SRSF1 from the cytoplasm to

nuclear speckles, whereas CLK1 phosphorylates RS2,

leading to broad nuclear dispersion of the SR protein

Mechanism of RS domain

phosphorylation

Although it is not uncommon for protein kinases to

exhibit somewhat relaxed substrate specificities and

phosphorylate more than one site in their protein target

[49–52], SRPK1 possesses the distinct ability to

effi-ciently insert numerous phosphates in close proximity

in RS domains In general, protein kinases recognize local charges flanking the site of phosphorylation [53] Random library searches have shown that SRPK pre-fers to phosphorylate serine, but not threonine, that is next to arginines [54] These studies were performed with a biased peptide library (arginine fixed in the P-3 position), that contained a single serine for modifi-cation In contrast, SRPKs phosphorylate many con-secutive serines in a richly electropositive substrate To accomplish this task, SRPK would need to maneuver deftly through a substrate whose charge is dramatically changing after each round of phosphorylation The question that this raises is whether these splicing kinases must re-engage the RS domain after each phosphoryla-tion reacphosphoryla-tion through a sequence of dissociaphosphoryla-tion–associ- dissociation–associ-ation steps (distributive phosphoryldissociation–associ-ation), or whether the RS domain can stay attached during subsequent rounds of phosphorylation, simply translating through the active site (processive phosphorylation) (Fig 3A) There are examples of protein kinases that catalyze multisite phosphorylation using either mechanism and sometimes a combination of both For example, the nonreceptor protein tyrosine kinase Src phosphorylates

up to 15 tyrosines in the protein Cas by a processive mechanism [49,50] In contrast, the dual specific protein kinase MEK activates MAPK through a two-site phos-phorylation mechanism that is fully distributive [52,55] Finally, the yeast cyclin–CDK complex from budding yeast (Pho80–Pho85) appears to phosphorylate five serines in the transcription factor Pho4, by a semipro-cessive mechanism [56]

The question of how a splicing kinase modifies an

SR protein was originally addressed for SRPK1 and its substrate SRSF1, with a start-trap protocol [57] In this experiment, a peptide inhibitor or a kinase-dead form

of SRPK1 (kdSRPK1) is added at the start of the reac-tion to a preformed enzyme–substrate complex in single turnover experiments (i.e [SRPK1] > [SRSF1]) If the enzyme phosphorylates the RS domain in a distributive manner, then free enzyme and phospho-intermediates

of the SR protein will be generated during the reaction that can be trapped by the inhibitor or kdSRPK1 and lead to reaction inhibition [47,48,57,58] However, if the mechanism is processive, then no free enzyme or phospho-intermediates will be released, and the peptide inhibitor or kdSRPK1 will not be able to stop the reac-tion For SRSF1, it was found that SRPK1 phosphory-lates, on average, five to eight of the 12 available serines in RS1, using a processive reaction before the enzyme dissociates and continues in a distributive man-ner These findings suggest that SRPK1 may use a dual-track mechanism, incorporating both processive and distributive phosphorylation steps (Fig 3A) Such

a process is expected to require a stable

enzyme–sub-strate complex Indeed, competition and single turnover

analyses indicate that the SRPK1–SRSF1 complex

dis-plays unusually high affinity, with a Kdbetween 50 and

100 nm [48,57] It is likely that this initial high affinity

is diminished during subsequent phosphorylation steps,

driving a shift from processive to distributive

phos-phorylation Accordingly, it has been shown that

SRPK1 inefficiently pulls down phosphorylated

SRSF1, whereas the unphosphorylated SR protein is

robustly pulled down [29,59] Although this mechanism

has been established with the use of SRSF1 that has a

rather short RS domain, it remains to be seen whether

processivity is a general feature of SRPKs and other

SR proteins with much larger RS domains It is

inter-esting to note that expanding the number of Arg-Ser

repeats in SRSF1 leads to enhanced processivity,

sug-gesting that other SR proteins could also be

phosphor-ylated by this mechanism [60]

Directional phosphorylation of RS

domains

Although SRPK1 can processively phosphorylate

sev-eral serines in SRSF1, it is not clear how this enzyme

attaches phosphates in close succession to a highly

charged substrate DNA polymerase, a classic

proces-sive enzyme, adds nucleotide triphosphates in a rigid

5¢ fi 3¢ direction, and initiates strictly at a DNA

pri-mer [61] To investigate whether SRPK1 is likewise

directional, an engineered protease footprinting tech-nique was employed [58] In these experiments, a lysine

is placed in the center of RS1 of SRSF1, and several additional Lysfi Arg mutations in RRM2 are then inserted When the resulting substrate is cleaved with LysC, two fragments easily identified on a gel can be obtained that correspond to the N-terminal and C-ter-minal halves of RS1 This method permits a fast and quantitative method for sorting phosphates placed on either the N-terminal or C-terminal end of RS1 By monitoring of the phosphorylation reaction in single turnover mode and conversion of the substrate into N-terminal and C-terminal fragments with LysC at various reaction stages, it can be shown that SRPK1 phosphorylates RS1 in a C-terminal to N-terminal direction (Fig 3B) Furthermore, by alteration of the position of the cleavage site in the RS domain, the ini-tiation region at the C-terminal end of RS1 (iniini-tiation box) can also be identified Interestingly, although SRPK1 prefers to start phosphorylation in the initia-tion box (Ser221–Ser225), mutainitia-tions in this region do not halt catalysis, indicating that the enzyme possesses the flexibility to move to other sites [58] This adapt-ability is likely to be an important feature of SRPK1 function, as the RS domains in other SR proteins are larger and more diverse (Fig 1) In addition to rapid RS1 phosphorylation, SRPK1 is capable of phosphor-ylating about three serines in RS2, although about 100-fold more slowly than the serines in RS1 [60] This overwhelming specificity for RS1 over RS2 is a result

SRPK1 SRSF1

Processive phosphorylation

Distributive phosphorylation

Directional phosphorylation

A

B

Fig 3 Mechanism of SRSF1 phosphoryla-tion by SRPK1 (A) Dual-track mechanism Start-trap analyses indicate that SRPK1 can phosphorylate up to eight serines in RS1, using a processive mechanism in which the kinase stays attached to the substrate after each round of phosphorylation The remain-ing serines in RS1 are modified in a distribu-tive manner, in which the kinase and substrate dissociate after each phosphoryla-tion event (B) Direcphosphoryla-tional phosphorylaphosphoryla-tion Mapping studies show that SRPK1 is a directional kinase that initially binds to an ini-tiation box (Ser221–Ser225) in the center of the RS domain, and then moves in an N-ter-minal direction to maximally phosphorylate RS1 The bold and light arrows indicate that processivity is progressively diminished as SRPK1 translates from the C-terminus to the N-terminus and dissociation becomes favored over forward catalysis.

of SRPK1’s preference for long Arg-Ser repeats, as

adding such repeats greatly increases phosphorylation

rates in RS2 These findings suggest that SRPK1 scans

RS domains in a search for long Arg-Ser stretches,

and is clearly capable of docking at additional sites on

the basis of local sequence factors Overall, SRPK1

moves in a well-defined C-terminal to N-terminal

direction along the RS domain of SRSF1, and possibly

could use a similar mechanism for other SR proteins,

although it may be capable of recognizing different

and, possibly, multiple initiation boxes

Docking interactions guide multisite

phosphorylation

Studies on the SRPK1-dependent phosphorylation of

SRSF1 have uncovered a remarkable catalytic

mecha-nism, displaying very unusual features How SRPK1

achieves multisite and directional phosphorylation at

the molecular level has recently been revealed through the X-ray structures of SRPK1 bound to either a short peptide substrate (Fig 2A) or the core region of SRSF1 (RRM2-RS1) (Fig 4A) These two structures show that SRPK1 possesses a docking region in the large lobe that can accept a portion of the RS domain This acidic docking groove in the kinase accommo-dates basic peptides about six to seven residues in length Mutation of several acidic residues within the docking groove (e.g Asp564, Glu571, and Asp548) eliminates processive phosphorylation and strong directional preferences within the RS domain [48] The peptide-bound form of SRPK1 allowed identification

of a small segment preceding the RS domain of SRSF1 [(RVKVDGPR(191–198)] as the cognate substrate site that specifically interacts with the docking groove Mutations of two basic residues in this segment (R191A and K193A) altered the catalytic mechanism, suggesting the importance of this region in SR protein

A

B

Fig 4 Model describing how the RS

domain of SRSF1 is threaded into the active

site of SRPK1 (A) X-ray structure of the

SRPK1–SRSF1 complex SRSF1 retained the

central RRM2 and RS1 segments and

lacked RRM1 and RS2 Only a portion of

RS1 is well defined in the complex (N¢-RS1,

residues 204–210), and resides in the

elec-tronegative docking groove The dotted

cir-cles present the possible path of the

segment of SRSF1 disordered in the crystal

from N¢-RS1 to RRM2 and the active site.

The surface rendition of CLK1 is shown in

the right panel The dotted circles represent

a possible path of the p-RS1 peptide

sub-strate on the kinase (B) Feeding

mecha-nism The N-terminal portion of RS1

(N¢-RS1) initially binds in the docking groove,

and the C-terminal portion (initiation box)

occupies the active site Representative

Arg-Ser pairs in both segments are

repre-sented as green hexagons The dotted line

represents intervening Arg-Ser-rich regions

in RS1 In the presence of ATP, RS1 is

phosphorylated in a C-terminal to N-terminal

direction until residues 191–198 (b4 of

RRM2) occupy the docking groove

Electro-positive side chains from the P+2 pocket

stabilize the phosphates on RS1.

phosphorylation [40] However, a subsequent structure

that cocrystallized with a truncated form of SRSF1

(RRM2-RS1) revealed that the N-terminal part of the

RS domain rather than residues 191–198 was bound to

the docking groove (Fig 4A) This was surprising, as

this RS segment [N¢-RS1; SYGRSRSRSR(201–210)],

binds to a pocket far from the active site (Fig 4A),

but eventually undergoes phosphorylation, as

deter-mined by mapping studies [58] These two kinase

struc-tures appeared to offer differing perspectives on which

regions outside the RRMs bind in the docking groove

In the RRM2-RS1-bound structure, the docking

groove binds an N-terminal segment of RS1

(resi-dues 201–210), whereas in the peptide-bound structure,

the docking groove binds sequences that are more

N-terminal from N¢-RS1 (residues 191–198)

As prior mapping studies showed that SRPK1

moves along the RS domain in a C-terminal to

N-ter-minal direction (Fig 3B), it is possible that the

struc-ture of the SRPK1–SRSF1 complex changes as a

function of phosphorylation, and that the two X-ray

structures present two distinct states along the catalytic

pathway This model was tested with mutant forms of

SRPK1 and SRSF1 that differentially cross-link as a

function of ATP A cysteine placed in the docking

groove of SRPK1 (K604C) cross-links with a cysteine

substituted in the segment preceding the RS domain

(K193C) only in the presence of ATP In comparison,

a second mutant form of SRSF1 in which a cysteine is

inserted in N¢-RS1 (R204C) cross-links with the dock-ing groove cysteine in the absence of ATP When con-sidered in light of the directional phosphorylation mechanism, these structural observations can be used

to propose a model for substrate phosphorylation in which the Arg-Ser repeat motif constitutes a mobile docking element, where the part of RS1 that is to be phosphorylated (N¢-RS1; residues 204–210) first serves

as a docking sequence placing a C-terminal serine from the initiation box at the active site (Fig 4B) As each serine undergoes phosphorylation, the docking motif moves by two residue increments towards the N-termi-nus Each Arg-Ser tract from the docking groove is sequentially displaced by an N-terminal tract with the originally identified docking motif in the docking groove at the end of the reaction In essence, the entire RS1 motif is fed through the active site of the kinase until the furthest N-terminal docking motif (resi-dues 191–196) ‘hits’ the kinase docking groove Inter-estingly, residues 191–196 lie in b-strand 4 of RRM2,

so that it must unfold in order to occupy the docking groove, a result supported by CD and mutagenesis experiments [28,62] Although the C-terminal residues

of RS1 are poorly defined in the structure, a single phosphoserine resulting form a small impurity in the cocrystallized nucleotide analog (AMPPNP) was found

in the basic P+2 pocket of the kinase (Fig 4B) Muta-tions in this pocket (R515A, R518A, and R561A) reduce the rate of phosphate incorporation into the

Fig 5 Surface electrostatic properties of SRPKs and CLKs Ribbon (top) and electro-static surface presentations (bottom) for Sky1p, SRPK1 and CLK1 are displayed All three molecules were crystallized as trun-cated proteins The nonconserved N-termi-nal and spacer domains were deleted in Sky1p and SRPK1 The N-terminal RS domain was deleted in CLK1.

terminal portion of RS1 [48], suggesting that the P+2

pocket stabilizes the growing phosphorylated RS

domain

Although structural studies on SRPK1 are the most

advanced at this time, it is likely that other

SR-direc-ted protein kinases will use aspects of the above

‘feed-ing’ mechanism For example, the yeast SRPK, Sky1p,

contains a similar charged docking groove to that of

SRPK1, which plays a role in the recognition of its

cognate substrate Npl3 (Fig 5) Although Npl3 lacks

a classic RS domain, it has a single RS dipeptide at

the very C-terminus of its RGG (Arg-Gly-Gly-rich)

domain In vitro studies on Sky1p and Npl3 have

shown that the RGG domain contains multiple

dock-ing motifs, at least one of which is essential for the

interaction of Npl3 with Sky1p [63] Although Sky1p

modifies a rather distinct substrate as compared with

SRSF1, it appears that the mobile docking element

may be a conserved feature in SR and SR-like

pro-teins and their kinases In comparison with SRPK1,

the X-ray structures of the CLKs revealed no deep

groove that would fit a peptide with geometric

com-plementarity (Fig 4A) Moreover, the corresponding

segment that would constitute the SRPK1 docking

groove is shallow and dispersed, with both acidic and

basic charge patches (Fig 5) This is comparable to

the highly acidic nature of the SRPK1 docking

groove This charge distribution suggests that the

hypo-phosphorylated RS domain with alternate positive

and negative charges could interact with CLK with

high efficiency as compared with the

unphosphory-lated RS domain That is, the product of SRPK1

phosphorylation might be the substrate of CLK We

showed that CLK1 will readily phosphorylate

approx-imately seven serines in RS2 in SRSF1 when it is

prephosphorylated in RS1 by SRPK1 [47] In

compar-ison, SRPK1 can phosphorylate about three serines in

RS2 but very inefficiently [60] The differences

between SRPK1 and CLK1 are likely to be rooted in

differences in docking elements and charge dispersal

(Fig 5) Whereas SRPK1 catalyzes a very strict,

directional mechanism, owing to its electronegative

docking groove, CLK1 lacking such a groove

ran-domly phosphorylates the RS domain of SRSF1 [29]

Our understanding of how CLKs modify RS domains

will be greatly advanced with the generation of a

CLK:RS domain structure and further investigations

into its substrate specificity

Conclusion

Recent structural and mechanistic studies on the

splic-ing kinase SRPK1 have uncovered a novel

phosphory-lation mechanism, in which a long section of the substrate’s RS domain is fed into the active site through a docking groove in the large lobe (Fig 4) This mechanism has similarities to polymerase-type chain reactions, where the enzyme binds in a defined region and then proceeds in a directional manner SRPK1 starts in a narrow initiation box that is defined

by the length of a greater binding channel encompass-ing the dockencompass-ing groove and the active site, a total dis-tance that can accommodate RS1 of the SR protein SRSF1 After initiation, the driving force for the direc-tional reaction is likely to involve a combination of repulsive interactions between the phosphoserines and the electronegative channel, and attractive electrostatic interactions between the phosphoserines and an elec-tropositive P+2 pocket Whether discrete initiation and extension reactions like those found in SRSF1 are common within the SR protein family awaits further investigations Although SRSF1 is a prototype for the family and the first to be investigated at a refined mechanistic level, it possesses a relatively small RS domain as compared with others in the SR protein family It will be interesting to learn how the catalytic principles uncovered for SRSF1 apply to SR proteins with considerably larger RS domains with multiple, lengthy Arg-Ser repeats

Acknowledgements This work was supported by NIH grants to J A Adams (GM67969) and G Ghosh (GM084277) References

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