The EMBO Journal Vol.18 No.11 pp.2923–2929, 1999
The crystal structure of the Physarum polycephalum
actin–fragmin kinase: an atypical protein kinase with
a specialized substrate-binding domain
Stefan Steinbacher1, Peter Hof,
Ludwig Eichinger2, Michael Schleicher2,
Jan Gettemans3, Joe ¨l Vandekerckhove3,
Robert Huber and Jo ¨rg Benz4
Abteilung Strukturforschung, Max-Planck-Institut fu ¨r Biochemie,
Ludwig-Maximilians-Universita ¨t, 80336 Mu ¨nchen, Germany and
3Flanders Interuniversity Institute of Biotechnology,
University of Gent, 9000 Gent, Belgium
4Present address: Department fu ¨r Chemie und Biochemie,
Universita ¨t Bern, 3012 Bern, Switzerland
Coordinated temporal and spatial regulation of the
actin cytoskeleton is essential for diverse cellular pro-
cesses such as cell division, cell motility and the forma-
tion and maintenance of specialized structures in
differentiated cells. In plasmodia of Physarum poly-
cephalum, the F-actin capping activity of the actin–
fragmin complex is regulated by the phosphorylation
of actin. This is mediated by a novel type of protein
kinase with no sequence homology to eukaryotic-type
protein kinases. Here we present the crystal structure
of the catalytic domain of the first cloned actin kinase
in complex with AMP at 2.9 Å resolution. The three-
dimensional fold reveals a catalytic module of ~160
residues, in common with the eukaryotic protein kinase
superfamily, which harbours the nucleotide binding
site and the catalytic apparatus in an inter-lobe cleft.
Several kinases that share this catalytic module differ
in the overall architecture of their substrate recognition
domain. The actin–fragmin kinase has acquired a
unique flat substrate recognition domain which is
supposed to confer stringent substrate specificity.
Keywords: actin phosphorylation/crystal structure/
cytoskeleton reorganization/fragmin/protein kinase
Protein phosphorylation plays a key role in regulating the
dynamic rearrangements of the cytoskeleton starting with
upstream signalling cascades (Eby et al., 1998) resulting
in phosphorylation of downstream effectors which directly
interact with actin or tubulin. For example, the signalling
pathway of Rac-mediated stimulus-induced actin reorgan-
ization results in phosphorylation of cofilin by LIM-kinase
(Arber et al., 1998) which abolishes cofilin’s actin binding
and depolymerization activities (Arber et al., 1998; Yang
et al., 1998). Phosphorylation of actin itself has been
observed repeatedly. Treatment of mammalian cells with
epidermal growth factor induces rapid phosphorylation of
actin in the cortical skeleton (van Delft et al., 1995).
© European Molecular Biology Organization
Stage-dependent phosphorylation of actin at Tyr53 in
Dictyostelium discoideum (Howard et al., 1993; Jungbluth
et al., 1995) is associated with morphological alterations
and reorganization of the actin cytoskeleton.
Plasmodial fragmin from the slime mould Physarum
polycephalum is a member of the gelsolin family which
has been implicated in cellular processes that require rapid
actin cytoskeleton reorganization, and interferes with the
growth of F-actin by severing actin filaments and capping
their barbed ends. The EGTA-resistant 1:1 complex
between actin and fragmin has been identified as the sole
in vivo target for a specific protein kinase (actin–fragmin
kinase, AFK) that phosphorylates actin mainly at Thr203
and to a minor extent at Thr202 in the actin–fragmin
complex but not in G-actin (Gettemans et al., 1992;
De Corte et al., 1996). The phosphorylation sites are
located at the minor contact site for DNase I (Kabsch
et al., 1990) and at one of the proposed actin–actin contact
sites along the long-pitch helix of F-actin (Holmes et al.,
1990). The F-actin nucleating activity of actin–fragmin is
abolished upon phosphorylation in vitro and its capping
activity becomes Ca2?-dependent. The latter observ-
ation was corroborated by microinjection of the (un)phos-
phorylated actin–fragmin complex in mammalian cells
(Constantin et al., 1998). These data indicate that actin
polymerization in Physarum can be controlled by actin
phosphorylation in a Ca2?-dependent manner.
Biochemical studies and cloning of the AFK resulted
in the identification of an 80 kDa protein representing a
novel type of protein kinase (Eichinger et al., 1996). Two
domains of ~35 kDa are linked by a sequence stretch of
50amino acidsrich inproline andserineresidues. Whereas
the C-terminal part harbours six so-called ‘kelch’-repeats,
indicating a six-bladed propeller structure (Bork and
Doolittle, 1994), the N-terminal part consists of a cata-
lytically active protein kinase domain (cAFK) which,
most notably, does not show any sequence similarities to
eukaryotic protein kinases and lacks all signature motifs
that characterize this super-family (Hanks and Hunter,
1995; Eichinger et al., 1996). Here we report on the
crystal structure of cAFK that was solved to gain insight
structural relationships to typical protein kinases.
Results and discussion
Recombinant cAFK was crystallized from high salt con-
ditions in the presence of AMP. Other nucleotides or
nucleotide analogues did not give crystals suitable for
structure determination. The structure was solved by single
isomorphous replacement and two-fold non-crystallo-
graphic symmetry averaging. The structure has been
refined to a crystallographic R-factor of 19.9% and Rfree
S.Steinbacher et al.
Table I. Data collection, phasing and refinement statistics
Rfree(%) r.m.s. bond
r.m.s. bonded B
NATI5148 22 28919.827.2 0.0111.82 3.430.31
NATI: 15.0–2.9 Å (3.06–2.90 Å); PCMB 15.0–3.3 Å (anomalous completeness). PCMB: 4 mM p-chloromercuribenzoic acid, 18 h.
Rsym? Σ|I–?I?|/ΣI, where I ? observed intensity, ?I? ? average intensity from multiple observation. Riso? Σ||FPH|–|FP||/Σ|FP|, where |FP| ?
protein structure factor amplitude, |FPH| ? heavy-atom derivative structure factor amplitude. Phasing power ? r.m.s. (|FH|/E), where FH? heavy-
atom structure factor amplitude and E ? residual lack of closure.
RC? Σ|FPH? FP– FHcalc|/Σ|FPH? FP| (centric reflections).
of 27.2% using data in the resolution shell from 8.0 to
2.9 Å (see Materials and methods; Table I). The model
incorporates residues 2–343 and one AMP molecule;
residues 33–47 are not defined by electron density. cAFK
has overall dimensions of ~30?50?60 Å and is comprised
of eight β-strands (S1–S8) and 11 α-helices (H1–H11),
organized in two lobes (Figure 1). The 165-residue
N-terminal lobe consists of four α-helices (H1–H4) and
a five-stranded, anti-parallel β-sheet (S1–S5) of topology
1-2-3-5-4. Helices H3 and H4 are inserted between
β-strands S4 and S5, and helix H2 is between S3 and S4.
The β-strand S1 is preceded by 72 residues that wrap
around the back side of the N-lobe with a loop structure
that is located at the side entrance of the nucleotide-
binding cleft and connects the N- and C-lobe, including
14 disordered residues. Helices H3 and H4 pack against
the β-sheets of the N-lobe. The N-terminal helix H1 packs
against H2 of the N-lobe and H10 of the C-lobe. A hinge
region at the bottom of the nucleotide binding-cleft is
located between β-strands S5 and S6 and connects the
N- and C-lobe. The 178-residue C-terminal lobe consists
of the remaining seven α-helices (H5–H11) and a three-
stranded, anti-parallel β-sheet (S6–S8) of topology 6-7-8.
Helices H5 and H6 are inserted between β-strands S6 and
S7. β-strand S8 is followed by helices H7–H11 which
contact helices H1, H2, H5 and H6, completing an arch
of nine α-helices that surrounds one side of the nucleotide-
binding cleft and packs against the β-sheet of the C-lobe.
Conserved catalytic core
Comparison of cAFK to the catalytic subunit of cAMP-
dependent protein kinase (cAPK) (Knighton et al., 1991;
Bossemeyer et al., 1993) as a prototype of eukaryotic
protein kinases reveals a topologically equivalent region
which is common to both kinases (Figures 1 and 2). This
region spans residues 73–232 in cAFK and includes the
secondary structure elements from S1 to S8. Helices H2
and H6 correspond to helices C and E in cAPK. This
corresponds to subdomains 1–7 in eukaryotic protein
kinases and comprises a minimal kinase module that
contains all elements necessary for phosphoryl transfer.
The cAFK and cAPK structures diverge C-terminal to
structure-based sequence comparison results in a sequence
identity as low as 14% using 76 topologically equivalent
residues out of 159 residues of the subdomains in common.
The highest local similarity is observed for the hinge
region between N- and C-lobe (cAFK: MELVRG; cAPK:
MEYVPG) although this region does not represent a
signature sequence in typical protein kinases. In particular,
the signature sequences (Hanks and Hunter, 1995) H/Y-
R-D-L/I-K-P-X-N for Ser/Thr kinases, H-R-D-L-R/A-A-
A/R-N for tyrosine kinases of the catalytic loop, G-X-G-
X-X-G of the glycine rich loop or the well known DFG
motif preceding the activation segment are not observed
in cAFK. These regions deviate structurally from all other
known protein kinases. The glycine rich loop connecting
β-strands S1 and S2 is shorter by one residue in cAFK.
The catalytic loop in cAFK that ranges from Asp204 to
Asn218 (corresponding to Asp166 and Asn171) bears an
insertion of nine residues providing contacts with helices
H8 and H9, and is therefore of structural importance, but
it also protrudes from the otherwise flat surface.
Weak autophoshorylation has been observed for recom-
binant AFK (Eichinger et al., 1996), but it is unknown
whether the region corresponding topologically to the
activation loop has an analogous function. There are two
serine residues, Ser233 and Ser241, in the coil region
candidates for autophosphorylation. The distance of nine
residues between the beginning of the activation segment
and Ser241 would correspond to that observed in other
kinases (Hanks and Hunter, 1995). High temperature
factors are observed for the residue range from Ile236 to
Arg249, which involves the activation segment and the
N-terminal half of helix H7 with the highest temperature
factors being found in both asymmetric monomers around
Ser241. This indicates potential flexibility compatible with
regulatory functions for this region.
Superimposing the adenine moiety in cAFK and cAPK
reveals an analogous mode of nucleotide binding. The
adenine is located between Ile96 from β-sheet S3 and
Met220 from S7, and occupies a mostly hydrophobic
pocket between both lobes. Other residues include Leu85,
Phe87, Pro121, Met162 and Ile231 with the connecting
hinge region at the base of the cleft. Polar van der Waals
contacts are made with the backbone in the hinge region
with a hydrogen bond of the N6 amino group to the
carbonyl group of Glu163. The ribose forms only van der
Waals contacts to Oγof Ser78 of the N-lobe, and to the
Crystal structure of actin–fragmin kinase
Fig. 1. (A) Ribbon diagram of the catalytic domain of actin–fragmin kinase (cAFK). View into the nucleotide-binding cleft with bound AMP
depicted in green. The N-lobe is located above, the C-lobe below this cleft. Helices are labelled H1–H11, β-strands S1–S8. The catalytic kinase
module, in common with eukaryotic-type protein kinases (ePKs), is coloured in light blue. Parts of the kinase module that differ from ePKs are
shown in dark blue, including helices H3 and H4 and the insertion into the catalytic loop. Parts that topologically differ from ePKs are shown in red.
(B) Perpendicular view to (A) as seen from the right side. (C) Sequence and secondary structure of cAFK. Conserved catalytic residues are shaded
in yellow, the glycine rich loop in magenta, a potential site of autophosphorylation in green. Colour coding of secondary structural elements as in (A).
S.Steinbacher et al.
Fig. 2. Crystal structure of cAPK with bound ATP (green) and PKI-inhibitor fragment (light green) for comparison. Orientation is with aligned
nucleotides in the kinase modules as in Figure 1A. Colour coding as in Figure 1. Helices are labelled A–J, β-sheets 1–9. The peptide inhibitor is
bound to a richly structured cleft with its N-terminal part. The main contacts involve helices D, F, G and H. These parts are absent in cAFK that has
a flat substrate recognition surface (compare with Figure 1).
side chain of Thr169 and the carbonyl group of Asp217
from the C-lobe. The glycine rich loop helps to position
the phosphate by formation of a hydrogen bond to the
backbone amide of Thr82, although it probably adopts an
arbitrary conformation in the presence of AMP, resulting
in high temperature factors and a less well-defined electron
density. This loop has been reported to be a relatively
flexible element in other kinases as well (Cox et al., 1994)
and its stability highly depends on bound ligands. The
same is true for the inter-lobe angle which indicates either
an open (inactive) or closed (active) state.
Five strictly conserved residues are found in the active
(Figure 3) (residue numbers for cAPK in parentheses):
Lys98 (72), Glu106 (91), Asp204 (166), Asn218 (171) and
Asp232 (184). Lys98 contacts the α-phosphate of AMP,
the corresponding residue in cAPK additionally contacts
the β-phosphate of the bound nucleotide. Lys98 forms a
conserved salt bridge with Glu106. The catalytic loop
harbours Asp204 and Asn218. Asp166 in cAPK interacts
with the incoming substrate and is thought to act as a
base required for deprotonation of the substrate hydroxyl
group enabling efficient hydrolytic attack at the γ-phos-
phate. Asn171 binds to a second divalent metal ion. The
α-phosphate of AMP makes contacts to Asp232. The role
of the analogous Asp184 of the DFG motif in cAPK
involves binding of the Mg2?ion that bridges the β- and
Specialized substrate-binding domain
The substrate-binding regions of typical protein kinases
are well established (Johnson et al., 1998), by inhibitor
binding in cAPK (Knighton et al., 1991; Bossemeyer
et al., 1993), and by pseudosubstrate-like binding in
twitchin kinase (Hu et al., 1994) or calcium/calmodulin-
dependent protein kinase I (Goldberg et al., 1996). The
N-terminal part of the substrate analogue is bound to a
channel on the surface of the protein that is created by
the edge of helix D along with an extensive region
stretching from the end of helix F through helices G and
H (Figure 2). The precise orientation of the substrate
analogue varies as it is observed for the orientation of
helix D. These structural elements belong to subdomains
5, 9 and 10 in the primary structure of typical protein
comprising the activation segment, appears to play a major
role in recognition of peptide substrates in cAPK. These
structural elements are absent in cAFK including helix D
that topologically belongs to the kinase module. Based on
the binding mode of the PKI fragment in the active site
cleft of the catalytic module of cAPK, the phosphorylated
loop around Thr203 in subdomain 4 of actin can be docked
into the active site cleft of cAFK and allows the prediction
of regions potentially important for substrate interaction.
A remarkable complementarity in shape thus emerges,
that allows docking of actin into the catalytic loop through
the cleft between subdomains 2 and 4 of actin, exposing
Thr211 and Glu213 of cAFK. The ‘activation segment’,
the C-terminus of helix H6 and the N-terminal half of
helix H7 of cAFK would contact the third β-strand (from
Leu65 to Lys68) at the edge of a β-pleated sheet of actin
subdomain 2 (Kabsch et al., 1990). Helix H8 of cAFK
would face the first helix (from Gly182 to Thr194) in
subdomain 4 of actin (Figure 4). The catalytic loop
insertion and the surrounding helices H6, H7 and H8
therefore probably form a relatively flat substrate recogni-
tion domain thatdiverges significantly from the elaborately
structured substrate recognition domain in eukaryotic
protein kinases (Figures 1 and 2). Since only the structure
of gelsolin segment-1 in complex with actin is known
(McLaughlin et al., 1993), where segment-1 binds to a
cleft between subdomains 1 and 3, the role of fragmin,
fragmin60 (Furuhashi et al., 1989, 1992) or Dictyostelium
severin in substrate recognition by cAFK remains unclear.
However, this structure shows that the phosphorylated
loop around Thr203 is considerably flexible in comparison
to the actin–DNase I complex, and this may explain why
neighbouring threonine residues in actin can also be
Crystal structure of actin–fragmin kinase
Fig. 3. Comparison of the actives site of cAFK (A) and cAPK (B). The orientation corresponds approximately to a 90° counterclockwise rotation
compared with Figure 1A. (A) The N-lobe is located on the left side, the C-lobe on the right side. Hydrophobic residues that form the adenine
binding pocket are in orange, bound AMP in green and strictly conserved catalytic residues compared with typical eukaryotic protein kinases in red.
The glycine rich loop contacting the α-phosphate is shown in magenta, (B) cAPK colour coding as in (A). The catalytic mechanism of phosphoryl
transfer by the kinase module appears to be strictly preserved throughout evolution, even in very distant relatives that have acquired completely
different substrate-binding modes.
Fig. 4. Proposed interaction of cAFK and actin. Actin coordinates were taken from the actin–DNase I complex (Kabsch et al., 1990). Subdomains
1–4 of actin are shown as wire, the orientation of cAFK is similar to Figure 1B. The phosphorylated loop of actin in subdomain 4 (coloured in red)
was docked into the active site, the phosphorylated residue Thr203 is shown as ball-and-stick model. Subdomain 2 of actin is predicted to interact
with helices H7 and H11 and the ‘activation segment’, subdomain 4 mainly with helices H6 and H8. The insertion into the catalytic loop of cAFK
(dark blue) is complementary to the cleft between subdomains 2 and 4 of actin and therefore probably essential for specific recognition of actin. As
gelsolin segment-1 binds to the cleft between subdomain 1 and 3 of actin, complete fragmin is expected to provide additional contacts between the
substrate complex (actin–fragmin) and AFK.
phosphorylated (Furuhashi et al., 1992; Gettemans
et al., 1992).
This analysis shows that the atypical actin–fragmin kinase
from P.polycephalum, which phosphorylates actin only
when actin is complexed with plasmodial fragmin, bears
a structural relationship to the eukaryotic-type protein
kinase family with respect to the kinase module. A clue
to the observed specificity can be deduced from the unique
to the inter-lobe cleft of actin. This substrate binding
domain discriminates AFK from the eukaryotic-type
Other divergent representatives of the protein kinase
kinases. The microbial aminoglycoside kinase APH(3?)-
IIIa catalyses the phosphorylation of a broad spectrum of
aminoglycoside antibiotics (Hon et al., 1997). Its structure
can roughly be superimposed in parts of the secondary
structure elements on subdomains 1–9, ranging from
β-sheet 1 to helix F in cAPK. Approximately the same
region is encountered in the type IIβ phosphatidylinositol
phosphate kinase (Rao et al., 1998), a lipid kinase with a
S.Steinbacher et al.
critical role in eukaryotic signal transduction pathways.
These structures demonstrate that a considerably smaller
part, the kinase module, is sufficient for binding ATP and
phosphorylating the substrate.
The structurally conserved character of features that are
essential for phosphoryl transfer, identified in a number
of protein kinases, suggests a stringently conserved mech-
kinase module. The conserved topology of the catalytic
core and the variability in substrate recognition observed
in cAFK, eukaryotic-type protein kinases, APH(3?)-IIIa,
and IIβ phosphatidylinositol phosphate kinase, suggest
divergent evolution from an ancestral kinase. Specific
requirements for substrate recognition as in AFK, or a flat
interface for membrane interaction as in phosphatidyl-
inositol phosphate kinase (Rao et al., 1998), have resulted
in specialized substrate-binding domains.
The pronounced role of eukaryotic-type protein kinases
in signal tranduction in highly specialized and differenti-
ated cells of multicellular organisms can be seen as the
major driving force underlying their enormous diversity.
As a result, they have evolved into one of the largest
protein families, comprising ~1% of the human genome
(Hanks and Hunter, 1995), but have retained most features
of substrate recognition surprisingly well. However, as
illustrated by the actin–fragmin kinase, substrate recogni-
tion can be much more diverse in true protein kinases than
may predict that other unusual protein kinases (Hanks and
Hunter, 1995) are also structurally related to the eukaryotic
protein kinase superfamily.
Materials and methods
Recombinant cAFK was expressed in Escherichia coli and purified as
described previously (Eichinger et al., 1996). The protein was dialysed
against 10 mM MES/NaOH pH 7.5, and concentrated to 10 mg/ml. For
crystallization 5 µl of the protein solution were mixed with 2 µl of a
10 mM stock solution AMP and 5 µl of 2.0 M Li2SO4in 100 mM MES/
NaOH pH 6.0, and equilibrated over 2.0 M Li2SO4. Trigonal crystals of
space group P3(2)21 with a ? b ? 178.9 Å, c ? 59.3 Å, α ? β ?90°
and γ ? 120° grew within 1 week to a maximum final size of 0.4 mm.
X-ray diffraction data were collected on a Mar research imaging plate
detector mounted on a Rigaku rotating anode X-ray generator, operating
at 50 kV and 100 mA. Measured intensities were integrated with
MOSFLM (Leslie, 1991) and scaled and merged using the CCP4
program suite (Collaborative Computational Project, 1994). A heavy
atom derivative was prepared by soaking a crystal in 4 mM of
p-chloromercuribenzoic acid for 18 h. Seven heavy atom sites were
identified with SHELXS (Sheldrick et al., 1993). Heavy atom parameters
were refined with MLPHARE (Collaborative Computational Project,
1994). The mean figure of merit was 0.34.
The 3.3 Å electron density was improved by solvent flattening (68%
solvent content) with DM (Collaborative Computational Project, 1994).
The NCS operators for two-fold non-crystallographic averaging were
derived from a partial model and improved with the programme
IMPROVE (Kleywegt and Jones, 1993). Cyclic averaging with RAVE
(Kleywegt and Jones, 1993) resulted in a back-transformation R-factor
Rback? 17.1%. The resulting electron density was of superb quality.
The complete model for one monomer and the AMP molecule could be
traced in the first round of model building with the programme FRODO
(Jones, 1978). The structure was refined with X-PLOR (Bru ¨nger, 1992).
A test set of 5% of the reflections was used for cross-validation. NCS
constraints were applied to the atomic positions (r.m.s.d. of 0.31 Å for
all atoms) but not to temperature factors as protomer A had a significantly
lower average B-factor (44.0 Å2) than protomer B (66.1 Å2). A bulk
solvent correction was applied in the final stages as implemented in
X-PLOR with |F|?2σ|F| in the resolution range 15.0–2.9 Å. The structure
displays good stereochemical parameters as estimated by the programme
PROCHECK (Laskowski et al., 1993) with 86.7% in the most favoured
region and 11.6 in additionally allowed regions in a Ramachandran plot
(Ramachandran and Sasisekharan, 1968).
The coordinates have been deposited with the Protein Data Bank with
the accession code 1cjg.
This work was supported by the Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 413 and the Fonds der Chemischen Industrie.
J.G. is a Postdoctoral Fellow of the Fund for Scientific Research-Flanders
Arber,S., Barbayannis,F.A., Hanser,H., Schneider,C., Stanyon,C.A.,
Bernard,O. and Caroni,P. (1998) Regulation of actin dynamics through
phosphorylation of cofilin by LIM-kinase. Nature, 393, 805–809.
Bork,P. and Doolittle,R.F. (1994) Drosophila kelch motif is derived from
a common enzyme fold. J. Mol. Biol., 236, 1277–1282.
Bossemeyer,D., Engh,R.A., Kinzel,V., Ponstingl,H. and Huber,R. (1993)
Phosphotransferase and substrate binding mechanism of the cAMP-
dependent protein kinase catalytic subunit from porcine heart as
deduced from the 2.0 Å structure of the complex with Mn2?adenylyl
imidodiphosphate and inhibitor peptide PKI(5–24). EMBO J., 12,
Bru ¨nger,A. (1992) X-PLOR version 3.1. A System for Crystallography
and NMR. Yale University Press, New Haven, CT.
Collaborative Computational Project, 4 (1994) The CCP4 suites:
programs for protein crystallography. Acta Crystallogr., D50, 760–763.
Constantin,B., Meerschaert,K., Vandekerckhove,J. and Gettmans,J.
(1998) Disruption of the actin cytoskeleton of mammalian cells by
phosphorylation and regulated by Ca2?ions. J. Cell Sci., 111,
Cox,S., Radzio-Andzelm,E. and Taylor,S.S. (1994) Domain movements
in protein kinases. Curr. Opin. Struct. Biol., 4, 893–901.
De Corte,V., Gettemans,J., Waelkens,E. and Vandekerckhove,J. (1996)
In vivo phosphorylation of actin in Physarum polycephalum. Study of
the substrate specificity of the actin–fragmin kinase. Eur. J. Biochem.,
Eby,J.J., Holly,S.P., van Drogen,F., Grishin,A.V., Peter,M., Drubin,D.G.
and Blumer,K.J. (1998) Actin cytoskeleton organization regulated by
the PAK family of protein kinases. Curr. Biol., 8, 967–970.
Gettemans,J. (1996) A novel type of protein kinase phosphorylates
actin in the actin–fragmin complex. EMBO J., 15, 5547–5556.
Furuhashi,K., Hatano,S., Ando,S., Nishizawa,K. and Inagaki,M. (1992)
Phosphorylation by actin kinase of the pointed end domain on the
actin molecule. J. Biol. Chem., 267, 9326–9330.
Furuhashi,K. and Hatano,S. (1989) A fragmin-like protein from
plasmodium of Physarum polycephalum that severs F-actin and caps
the barbed end of F-actin in a Ca2?-sensitive way. J. Biochem. (Tokyo),
Gettemans,J., De Ville,Y., Vandekerckhove,J. and Waelkens,E. (1992)
Physarum actin is phosphorylated as the actin–fragmin complex at
residues Thr203 and Thr202 by a specific 80 kDa kinase. EMBO J.,
Goldberg,J., Nairn,A.C. and Kuriyna,J. (1996) Structural basis for the
autoinhibition of calcium/calmodulin-dependent protein kinase I. Cell,
Hanks,S.K. and Hunter,T. (1995) The eukaryotic protein kinase
superfamily: kinase (catalytic) domain structure and classification.
FASEB J., 9, 576–596.
Holmes,K.C., Popp,D., Gebhard,W. and Kabsch,W. (1990) Atomic model
of the actin filament. Nature, 347, 44–49.
Hon,W.-C., McKay,G.A., Thompson,P.R., Sweet,R.M., Yang,D.S.C.,
Wright,G.D. and Berghuis,A.M. (1997) Structure of an enzyme
required for aminoglycoside antibiotic resistance reveals homology to
eukaryotic protein kinases. Cell, 89, 887–895.
Howard,P.K., Sefton,B.M. and Firtel,R.A. (1993) Tyrosine phos-
phorylation of actin in Dictyostelium associated with cell-shape
changes. Science, 259, 241–244.
is inhibited byactin
Crystal structure of actin–fragmin kinase
Hu,S.-H., Parker,M.W., Lei,J.Y., Wilice,M.C.J., Benian,G.M. and
Kemp,B.E. (1994) Insight into autoregulation from the crystal structure
of twitchin kinase. Nature, 369, 581–584.
Johnson,L.N., Lowe,E.D., Noble,M.E.M. and Owen,D.J. (1998) The
structural basis for substrate recognition and control by protein kinases.
FEBS Lett., 430, 1–11.
Jones,T.A. (1978) A graphics model building and refinement system for
macromolecules. J. Appl. Crystallogr., 11, 268–272.
Jungbluth,A., Eckerskorn,C., Gerisch,G., Lottspeich,F., Stocker,S. and
Schweiger,A. (1995) Stress-induced tyrosine phosphorylation of actin
in Dictyostelium cells and localization of the phosphorylation site to
tyrosine-53 adjacent to the DNase I binding loop. FEBS Lett., 375,
Kabsch,W., Mannherz,H.G., Suck,D., Pai,E.F. and Holmes,K.C. (1990)
Atomic structure of the actin DNase I complex. Nature, 347, 37–44.
Kleywegt,G.J. and Jones,T.A. (1993) Mask made easy. ESF/CCP4
Newsletter, 28, 56–59.
Knighton,D.R., Zheng,J.H., Ten Eyck,L.F., Ashford,V.A., Xuong,N.H.,
Taylor,S.S. and Sowadski,J.M. (1991) Crystal structure of the catalytic
subunit of cyclic adenosine monophosphate-dependent protein kinase.
Science, 253, 407–414.
Laskowski,R.A., MacArthur,M.W., Moss,D.S. and Thornton,J.M. (1993)
PROCHECK: a program to check the stereochemical quality of protein
structures. J. Appl. Crystallogr., 26, 283–291.
Leslie,A.G.W. (1991) Recent Changes to the MOSFLM Package for
Processing Film and Image Plate Data. SERC Laboratory, Daresbury,
Warrington WA44AD, UK.
McLaughlin,P.J., Gooch,J.T., Mannherz,H.-G. and Weeds,A.G. (1993)
Structure of gelsolin segment 1–actin complex and the mechanism of
filament severing. Nature, 364, 685–692.
Ramachandran,G.N. and Sasisekharan,V. (1968) Conformation of
polypeptides and proteins. Adv. Protein Chem., 23, 283–438.
Rao,V.D., Misra,S., Boronenkov,I.V., Anderson,R.A. and Hurley,J.H.
(1998) Structure of type IIb phosphatidylinositol phosphate kinase: a
protein kinase fold flattened for interfacial phosphorylation. Cell, 94,
Sheldrick,G.M., Dauter,Z., Wilson,K.S., Hope,H. and Sieker,L.C. (1993)
The application of direct methods of patterson interpretation to high-
resolution native protein data. Acta Crystallogr., D49, 18–23.
van Delft,S., Verkleij,A.J., Boonstra,J. and van Bergen en Henegouwen,
P.M.P. (1995) Epidermal growth factor induces serine phosphorylation
of actin. FEBS Lett., 357, 251–254.
Yang,N., Higuchi,O., Ohashi,K., Nagata,K., Wada,A., Kangawa,K.,
Nishida,E. and Mizuno,K. (1998) Cofilin phosphorylation by LIM-
kinase 1 and its role in Rac-mediated actin reorganization. Nature,
Received March 9, 1999; revised and accepted April 7, 1999