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

REVIEW ARTICLE
Phosphorylation mechanism and structure of
serine-arginine protein kinases
Gourisankar Ghosh
1
and Joseph A. Adams
2
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:
(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).

FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works 587
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 U2AF
35
) 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 feeding 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
PRSPSYGRSRSRSRSRSRSRSRSNSRSRSYSPRRSRGSPRYSPRHSRSRSRT-C′
SRSF4
316
SRSF6
160
SRSF7
118
SRSF5
88
SRSF2
105
SRSF3
79
SRSF1
50
RS1
RS2
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.
SRPK structure and mechanism G. Ghosh and J. A. Adams
588 FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works
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
(U2AF
35
) 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
G. Ghosh and J. A. Adams SRPK structure and mechanism
FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works 589

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 additional 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.
SRPK structure and mechanism G. Ghosh and J. A. Adams
590 FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works
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 Arg fi 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 truncation
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 reaction through a sequence of dissociation–associ-
ation steps (distributive phosphorylation), 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

G. Ghosh and J. A. Adams SRPK structure and mechanism
FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works 591
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 K
d
between 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¢fi3¢ 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 Lys fi 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 (initiation
box) can also be identified. Interestingly, although
SRPK1 prefers to start phosphorylation in the initia-
tion box (Ser221–Ser225), mutations 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) Directional phosphorylation.
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.

SRPK structure and mechanism G. Ghosh and J. A. Adams
592 FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works
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 (b4of
RRM2) occupy the docking groove. Electro-
positive side chains from the P+2 pocket
stabilize the phosphates on RS1.
G. Ghosh and J. A. Adams SRPK structure and mechanism
FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works 593
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 N-
Sky1p SRPK1 CLK1
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.
SRPK structure and mechanism G. Ghosh and J. A. Adams
594 FEBS Journal 278 (2011) 587–597 Journal compilation ª 2011 FEBS. No claim to original US government works
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 docking 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).
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