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INTRODUCTION |
The dynamic rearrangements of the actin cytoskeleton in motile
cells are mainly regulated by actin-binding proteins which either
interfere directly with the polymerization kinetics of actin or alter
the viscoelasticity of the filamentous network (for reviews, see
Refs.1-3). Severin from Dictyostelium discoideum, a model
for amoeboid cell motility, belongs to the class of F-actin fragmenting
and capping proteins whose members are structurally and functionally
related (4). This class includes among others the vertebrate proteins
gelsolin (5), villin (6), gCap39 (7), or from Physarum
polycephalum the protein fragmin (8). F-actin fragmenting proteins
are especially well suited for causing quick rearrangements in the
filamentous actin network. At micromolar Ca2+ levels they
sever actin filaments by rupturing the noncovalent bonds between actin
subunits in a filament. This leads to a rapid increase of short
filaments together with a dramatic decrease in viscosity. After having
severed the actin filaments, the proteins remain bound at the barbed
end of the filaments and thereby prevent filament elongation. It is
assumed that this results in solation of the viscous cytoplasm with a
large number of short but capped filaments. For several members of this
family it has been shown in vitro that uncapping is caused
by polyphosphoinositides. In vivo this could then lead to
free barbed ends ready for rapid elongation (9).
There is increasing evidence that actin fragmenting proteins might be
targets in signaling cascades to the cytoskeleton. Gelsolin has been
implicated in the phosphoinositide-mediated F-actin uncapping of human
platelets following stimulation of thrombin receptors (10). Fibroblasts
of gelsolin null mice have excessive actin stress fibers and migrate
more slowly than wild type fibroblasts (11), while overexpression of
gelsolin in NIH 3T3 fibroblasts leads to an increase in motility (12).
In addition to Ca2+ and polyphosphoinositides,
phosphorylation seems to play an important role in regulating proteins
from this family as well (13). However, except for a fragmin kinase
(14) no other kinase has been described in detail so far.
The intracellular responses to external signals are very often mediated
by kinase cascades. The best studied kinase cascade activated by
external signals is the mitogen-activated protein kinase
(MAPK)1 system. Its core
comprises a module of three kinases in which the most distal MAPK is
activated by a MAPK kinase (MAPKK) which itself is activated by a MAPKK
kinase (MAPKKK). MAPK modules are ubiquitous among eukaryotes and in
recent years it has become clear that in every cell several pathways,
responsive to different external stimuli, exist in parallel (15-17).
The protein kinase PAK1 from rat brain was identified based upon its
ability to interact with the small GTPases Rac1 and Cdc42. The binding
of active Rac1/Cdc42 stimulated autophosphorylation and activity of
PAK1. The sequence of PAK1 was found to be closely related to Ste20p, a
key regulator in the mating pheromone response pathway in
Saccharomyces cerevisiae, that acts via activation of a MAPK
cascade (18-20). The growing family of related kinases is referred to
as either the PAK or Ste20-like kinase family. Although their in
vivo role has not yet been clearly defined, PAK family members are
considered to be promising candidates for the mediation of both
Cdc42/Rac-induced effects, cytoskeletal reorganization, and
transcriptional activation via a MAPK cascade (21, 22).
Based on primary structure and mode of regulation, the PAK family can
be subdivided into two main branches. Close relatives of PAK1 and
Ste20p (true PAKs) are characterized by a C-terminal kinase domain and
an N-terminal regulatory domain of variable length that contains a
p21-binding domain (23) and, in some cases, a pleckstrin homology
domain as well. Members of the second branch of the PAK family, the
so-called GCK subfamily, have their catalytic domain positioned at the
N terminus followed by a C-terminal regulatory region (21). Here we
describe the isolation and characterization of a severin kinase from
Dictyostelium, whose 62-kDa subunit is most closely related
to human SOK-1, a member of the GCK subfamily of Ste20-like
kinases.
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MATERIALS AND METHODS |
Protein Purification--
Cells of D. discoideum
strain AX2 were cultivated axenically at 21 °C in 5-liter Erlenmeyer
flasks up to a density of 5 × 106 cells/ml, harvested
without starvation, and homogenized by nitrogen excavitation in a Parr
bomb essentially as described (24) in the presence of a mixture of
protease inhibitors in the homogenization buffer (25). Usually 50 to
80 g of cells (wet weight) from 12 liters of culture were used for
protein purification.
The 100,000 × g supernatant was adjusted to pH 8.0 and
loaded onto a DEAE cellulose (Whatman, Maidstone, United Kingdom) anion exchange column (2.5 × 10 cm) equilibrated with TEDABP, pH 8.0 (10 mM Tris/HCl, 1 mM EGTA, 1 mM
dithiothreitol, 0.02% NaN3, 1 mM benzamidine,
1 mM phenylmethylsulfonyl fluoride). After washing the
column with at least 2 column volumes of TEDABP, protein was eluted
with a linear salt gradient of 0-300 mM NaCl in TEDABP (400 ml). Under these conditions severin kinase eluted early in the
gradient between 40 and 80 mM NaCl (Fig. 1A).
All the following purification steps were performed with columns
connected to the BioLogic FPLC system (Bio-Rad, München,
Germany). The active fractions were pooled, the pH carefully adjusted
to 6.5, and loaded onto a S-Sepharose column (1 × 10 cm)
equilibrated with MEDABP, pH 6.5 (10 mM MES, 1 mM EGTA, 1 mM dithiothreitol, 0.02%
NaN3, 1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride). After washing, the column was developed
with a linear gradient of 0-400 mM NaCl (60 ml) in MEDABP
(Fig. 1B). Severin kinase eluted between 160 and 290 mM NaCl. The corresponding fractions were pooled, dialyzed against TEDABP, loaded onto a Mono Q HR 5/5 column (Pharmacia, Freiburg, Germany) equilibrated in the same buffer and bound protein eluted with a linear gradient of 0-400 mM NaCl (40 ml) in
TEDABP (Fig. 1C). The active fractions (250-320
mM NaCl) were pooled and solid ammonium sulfate was added
to 50% saturation. The resulting pellet was dissolved in 200 µl of
IEDANBP, pH 7.6 (10 mM imidazole, 1 mM EGTA, 1 mM dithiothreitol, 0.02% NaN3, 200 mM NaCl, 1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride) and loaded onto a Superose 12 HR 10/30
column (Pharmacia, Freiburg, Germany) equilibrated in the same buffer.
Severin kinase eluted at a position corresponding to a molecular mass
of approximately 300 kDa (Fig. 1D). Active fractions were
either used for biochemical characterization or for determination of
peptide sequences. Using this purification scheme we obtained about 10 µg of the 62-kDa polypeptide from 50 g of cells (wet weight).
During all purification steps the elution of severin kinase activity
was assayed by phosphorylation assays with either severin, the 1:1
actin-severin complex, or DS211C as a substrate (26). For size
calibration of the Superose 12 column the following proteins were used:
ferritin (450 kDa), catalase (240 kDa), aldolase (158 kDa), bovine
serum albumin (68 kDa), ovalbumin (45 kDa), chymotrypsin (25 kDa), and
cytochrome c (12.5 kDa; Fig. 1D, inset).
Rabbit actin was prepared from skeletal muscle according to Spudich and
Watt (27) and further purified by gel filtration on Sephacryl S300.
D. discoideum severin and actin were purified as described
(28). The concentration of actin was measured as described (29). All
other protein concentrations were determined by the method of Bradford
(30) using bovine serum albumin as a standard.
Recombinant regulatory domain of severin kinase was purified from
M15[pREP4] cells that had been transformed with the expression plasmid pQE32 containing the corresponding coding region. Cells were
grown at 37 °C, induced at an OD580 nm of 0.6 with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside for
2 h, harvested, opened by ultrasonication as described (28), and
insoluble material was pelleted (20 min, 30,000 × g).
The resulting pellet was stepwise re-extracted, once with TEDABP, pH
8.0, containing 150 mM NaCl and five times with TEDABP, pH
8.0, containing 6 M urea (TEDABUP). The latter five
supernatants were combined and applied onto a Ni-NTA column
equilibrated in the same buffer. The column was washed with TEDABUP and
then with TEDABUP containing 20 mM imidazole. The
regulatory domain was eluted with TEDABUP containing 200 mM imidazole. Regulatory domain purified in this way was slowly dialyzed against TEDABP, pH 8.0, and concentrated by ultrafiltration in a
Centricon-10 microconcentrator (Amicon GmbH, Witten, Germany). Two
rabbits were immunized with the recombinant protein according to
established procedures.
Immunoprecipitation--
Polyclonal antibodies directed against
the regulatory domain were used for immunoprecipitation of severin
kinase from either a partially purified fraction (severin kinase pool
after the Mono Q column) or from a crude fraction after opening the
Dictyostelium cells (100,000 × g
supernatant). Kinase containing fractions (700 µl) were incubated
overnight at 6 °C with 50 µl of polyclonal antibody 5196 or
polyclonal antibody 5197 in a total volume of 1 ml with a final
concentration of 0.1% Triton X-100 in phophate-buffered saline buffer
(150 mM NaCl, 100 mM
Na2HPO4, 30 mM
KH2PO4), pH 8.0. Control immunoprecipitations
were carried out in the absence of either polyclonal antibody or
severin kinase. Protein-A Sepharose beads (25 µl) were added, the
suspensions shaken for 1 h, and centrifuged for 2 min at
10,000 × g. The supernatants were removed, the pellets
washed three times with 300 µl of phosphate-buffered saline,
resuspended in 50 µl of TEDABP, pH 8.0, and aliquots (20 µl) used
in a phosphorylation assay with either severin or severin in the
actin-severin complex as a substrate. The remaining beads were boiled
after addition of 20 µl of 3 × SDS sample buffer and analyzed
by SDS-PAGE.
Phosphorylation Assays--
Severin kinase activity was assayed
in a reaction mixture (40 µl) containing 10 mM Tris/HCl,
pH 7.5, 1 mM dithiothreitol, 1 mM EGTA, 1 mM Na3VO4, 2 mM sodium
fluoride, 10 mM MgCl2, 0.05 mg/ml bovine serum
albumin, 0.1 mM ATP (2-5 µCi of [32P]ATP),
0.01% NaN3, and 1-4 µM substrate. The
reaction was initiated by addition of either the substrate or the
kinase to the reaction mixture and carried out at 30 °C. Severin
(2-4 µM final concentration), DS211C (2-4
µM), or the 1:1 actin-severin complex (1-2
µM each) were used as substrates. The actin-severin
complex was allowed to form in G-buffer, followed by the addition of
EGTA (1 mM final concentration) resulting in the EGTA
stable 1:1 complex (26). Determination of the substrate dependence of
severin kinase was carried out with different substrates at a final
concentration of 2 µM. For testing the Ca2+
dependence of the phosphorylation reaction, the mixture contained, in
addition, 2 mM Ca2+, and in the assays with
Mn2+-ATP, 10 mM MnCl2 instead of
MgCl2. The pH dependence was tested by the addition of 1/10
volume of the following buffers to the reaction mixture: 500 mM MES, pH 6.0 and 6.5, 500 mM MOPS, pH 7.0, 500 mM Tris/HCl, pH 7.5, 8.0, and 8.5. Activation by
autophosphorylation was tested by preincubating severin kinase with
unlabeled ATP for 0, 5, or 20 min in the absence of substrate, then
substrate was added and the reaction allowed to proceed for 20 min at
30 °C. If not stated otherwise phosphorylation was terminated after
30 min by the addition of 20 µl of 3 × concentrated SDS sample
buffer and boiling for 3 min. Proteins were separated by SDS-PAGE on minislab gels (110 × 83 × 0.5 mm) using the buffer system
of Laemmli (31). Electrophoresis was terminated before the running
front reached the lower buffer chamber and the gel was cut just above the running front to remove the lower gel strip which contains most of
the non-incorporated radioactive ATP. Protein bands were visualized by
staining with Coomassie Brilliant Blue and, after drying of the gels,
labeled proteins were detected by autoradiography on Kodak X-AR films.
For quantitation of incorporated phosphate, bands were scanned
densitometrically and intensities evaluated with the program NIH Image
1.61.
Cloning and DNA Sequence Analysis--
Tryptic fragments of the
62-kDa subunit of severin kinase were resolved by reversed-phase
chromatography and subjected to Edman degradation on an Applied
Biosystems gas-phase sequencer according to Eckerskorn et
al. (32). Four peptide sequences, K5 (EQQQQQQPTPV), K8
(PVAVQEQQQTP), K10 (GSFGEV), and K18 (TVAATTAPATTPASNAPT), were
obtained and used to construct degenerate PCR primers. PCR was
performed with genomic DNA of the AX2 strain as a template (denaturation: 94 °C, 60 s; annealing: 54 °C, 60 s;
elongation: 72 °C, 60 s; 25 cycles) with primers K8N
(5'-CGCGAATTCCCAGTHGCHGTHCAAGA-3') and K18C
(5'-CGCGGATCCGCTGGDGTDGTDGCWGG-3') and resulted in amplification of a
250-base pair genomic fragment. The translated open reading frame of
this fragment contained K5 as well as the appropriate amino acids
downstream of K8N and K18C. All primers contained 5'-overhang sequences
suitable for digestion with restriction enzymes; for degenerate
positions the following abbreviations are used: H = [A,T,C];
D = [G,A,T]; W = [A,T]. The 0.25-kb fragment generated
was labeled with [
-32P]dATP and employed to screen a
gt11 cDNA library (33) as described (34). From one positive
clone the cDNA insert was amplified by PCR using primers of the
gt11 flanking regions, cloned into pUC19 vector (35), and sequenced
with the chain termination dideoxy method (36) using uni and reverse
primers, as well as sequence specific oligonucleotide primers. The
isolated cDNA had a size of about 1.2 kb and contained the 3' end,
but lacked the 5' end of the gene. A 5' 0.8-kb EcoRI
fragment of this clone was used to screen a random primed
gt11
cDNA library (CLONTECH Inc., Palo Alto, CA)
which yielded another positive clone with an insert of about 1.3 kb
harboring the ATG start codon preceded by two in-frame stop codons but
lacking the 3' end of the gene. The two cDNA clones had about 1-kb
sequence in common and internal restriction sites were used to combine
the two clones to yield the full-length cDNA in pUC19. In order to
exclude possible errors resulting from PCR amplification of the
gt11
cDNA clones, we confirmed both sequences at least once with
independently amplified and cloned PCR products.
Standard techniques were used for cloning, transformation, and
screening (37). Searches for similarities to other protein sequences
were done with the program BLAST (38) using the combined non-redundant
entries of the Brookhaven Protein Data Bank, Swiss-Prot, PIR, and
GenBank at the NCBI. The sequence was analyzed by using the UWGCG
(University of Wisconsin Genetic Computer Group; Madison, WI) and
PHYLIP (Phylogeny Inference Package, version 3.5c by Joseph Felsenstein, University of Washington) program packages. Northern analysis followed established procedures (39).
The coding sequence for the regulatory domain (aa 277-478) was
amplified by PCR (denaturation 94 °C, 60 s; annealing 60 °C, 60 s; elongation 72 °C, 60 s; 25 cycles) with primers
Sevkin-Ntreg (5'-CGCGGATCCATATGAGAAGACAAAAATGGTTACAAT-3') and
Sevkin-Ct (5'-GCGAAGCTTTTATCTTTTAAGGGTTTCAATG-3').
Primer sequences corresponding to the coding sequence of severin kinase
are shown in italic and restriction sites in the 5' overhang sequence
for cloning in bold. The resulting PCR product was cloned into the BamHI, HindIII sites of the pQE32 expression
vector (Qiagen GmbH, Hilden, Germany). The complete expression
construct was sequenced to exclude possible errors resulting from the
PCR.
 |
RESULTS |
Partial Purification and Characterization of Severin
Kinase--
To identify protein kinases from D. discoideum
that phosphorylate cytoskeletal proteins, we screened DEAE column
fractions of soluble homogenates for kinase activities by adding actin, severin, or a 1:1 complex of both proteins as a substrate. We detected
an activity that phosphorylated the actin-fragmenting protein severin
either on its own or in a complex with actin. This severin kinase
activity was further purified by additional chromatographic steps
including gradient elution from S-Sepharose or Mono Q, and gel
filtration on Superose 12 (Fig. 1). In
the final Superose 12 gel filtration step the kinase eluted at a
position corresponding to a molecular mass of about 300 kDa (Fig.
1D, inset, Superose 12). These active fractions contained a
polypeptide of about 62 kDa which (i) coeluted with kinase activity,
(ii) was strongly phosphorylated in the presence of
[
-32P]ATP and (iii) shifted almost completely to a
higher molecular mass in SDS-PAGE after preincubation with unlabeled
Mg2+-ATP (Fig.
2A). The low percentage gel
(7.5% acrylamide) used in this experiment resolved the rather broad
62-kDa signal shown in Fig. 1 into at least three distinct bands (Fig.
2A, lane 3) which suggests multiple autophosphorylation of
the 62-kDa protein. It is not yet clear whether native severin kinase
is composed of only the 62-kDa subunit or whether it constitutes a
heteromer. Autophosphorylation of the 62-kDa polypeptide exactly
followed severin kinase activity during all purification steps and was therefore likely to represent the severin kinase (Fig. 1). In addition,
polyclonal antibodies directed against the regulatory domain
specifically precipitated the 62-kDa polypeptide from either a
partially purified severin kinase fraction or from the soluble fraction
after opening the Dictyostelium cells. In phosphorylation assays, the 62-kDa polypeptide in the immunoprecipitate as well as
added severin either on its own or in complex with actin were strongly
phosphorylated (Fig. 2B, lanes 2 and 3).
Phosphorylation of severin in the actin-severin complex was more
pronounced than phosphorylation of severin alone. Results obtained with
a second independently generated antiserum were very similar. Control
immunoprecipitations were carried out in the absence of either
polyclonal antibody or severin kinase fraction as described above. In
the absence of antibodies (Fig. 2B, lane 1) or severin
kinase (data not shown) there was no phosphorylation of severin. These
results clearly demonstrate that the 62-kDa polypeptide is essential
for severin kinase activity.

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Fig. 1.
Partial purification of severin kinase from
Dictyostelium cells. Cytosolic extract of
Dictyostelium was clarified at 100,000 × g
and then chromatographed on DEAE-cellulose (A). Peak
fractions of severin kinase (black horizontal bars) were
pooled and further purified on S-Sepharose (B) and Mono Q
columns (C). After ammonium sulfate precipitation (50%
saturation) and resolubilization, the severin kinase containing pool
was loaded onto Superose 12 (D). From this gel filtration
column severin kinase activity eluted early (black horizontal
bar) and calibration of this column with marker proteins indicated
that severin kinase had a molecular mass of about 300 kDa (D,
inset). The upper panels show the applied salt
gradients (open triangles) and protein elution (open
circles). Column fractions from each purification step were tested
in phosphorylation reactions for severin kinase activity with severin
in the actin-severin complex as a substrate. After phosphorylation
polypeptides were separated by SDS-PAGE in 12% gels, the gels dried,
and subjected to autoradiography (A-D, lower panels). The
positions of severin and of the 62-kDa subunit of severin kinase are
indicated. Please note the strong signal in the 62-kDa range that
exactly coelutes with severin kinase activity.
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Fig. 2.
Autophosphorylation of the 62-kDa subunit of
severin kinase (A) and specific immunoprecipitation of
severin kinase activity (B). A, the peak
fraction of severin kinase from the Superose 12 column was incubated
for 30 min at 30 °C in the absence (lane 1) or presence
of unlabeled ATP (lane 2), or with radioactive ATP
(lane 3). The proteins were separated by SDS-PAGE in 7.5%
gels, stained with Coomassie Blue (lanes 1 and
2), or stained and processed for autoradiography (lane
3). The positions of the 62-kDa polypeptide before (*) and after
(*') autophosphorylation are indicated. The presence of at least three
distinct bands in the autoradiogram suggests multiple
autophosphorylation of the 62-kDa polypeptide. B, partially
purified severin kinase from the Mono Q column was incubated in the
absence (lane 1) or presence of polyclonal antibodies
(lanes 2 and 3) that were raised against the
regulatory domain of the 62-kDa polypeptide. After precipitation with
protein-A Sepharose, the beads were used in phosphorylation reactions
with either severin (lanes 1 and 2) or the
actin-severin complex (lane 3) as substrates. Proteins were
separated by SDS-PAGE and processed for autoradiography as described
above. Please note that the specifically precipitated material showed
the characteristic autophosphorylation and phosphorylation of the
substrates.
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We used the peak fractions from the gel filtration column, the last
purification step, to biochemically characterize severin kinase and to
obtain sequences from tryptic peptides. Fig.
3 shows the time dependence
(A), the activation by autophosphorylation (B),
and the pH dependence (C) of severin kinase as measured by phosphorylation of domains 2 and 3 of severin (DS211C; see below). During early time points, there was an almost exponential increase of
incorporation of phosphate into the substrate which could be attributed
to self-activation of the kinase and excess of substrate (Fig.
3A). Autophosphorylation for 5 min increased the activity of
severin kinase more than 3-fold and a nearly 6-fold increase in
activity was observed after 20 min of in vitro
autophosphorylation (Fig. 3B). The activity of severin
kinase decreased rapidly at pH values below pH 7.0, while pH values
above 7.5 decreased its activity only moderately (Fig. 3C).
Routinely, phosphorylation assays were carried out for 30 min at pH 7.5 and 30 °C.

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Fig. 3.
Time dependence (A), activation
by autophosphorylation (B), and pH dependence of severin
kinase activity (C). The phosphorylation reactions
with DS211C as a substrate were carried out for the indicated periods
of time (A), for 20 min after allowing severin kinase to
autophosphorylate for 0, 5, or 20 min in the presence of unlabeled ATP
(B), or for 30 min at the pH values stated (C).
The reaction mixtures were separated by SDS-PAGE in 12% gels and the
dried and stained gels subjected to autoradiography. The radioactive
DS211C bands were scanned densitometrically and intensities evaluated
with the program NIH image 1.61.
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The substrate specificity of severin kinase has been tested with domain
1 (DS151), domain 2 (DS111M), and domains 2 and 3 (DS211C) of severin
(26), the 1:1 actin-severin complex, and with the
Dictyostelium actin-binding proteins
-actinin, ABP120 gelation factor, and hisactophilin. Besides severin, on its own as well
as in the 1:1 complex with actin, only DS151 (residues 1-151 of
severin) and DS211C (residues 152-362 of severin) turned out to be
substrates of severin kinase (data not shown). In particular DS211C was
very strongly phosphorylated being an even better substrate than native
severin. Since DS151 and DS211C have no overlapping amino acids,
severin kinase must either phosphorylate native severin at two or more
sites, or alternatively, there must be a cryptic phosphorylation site
in the constructs that is not accessible in the native molecule.
Severin phosphorylation appeared to be regulated also at the substrate
level as incorporation of phosphate was strongly reduced in the
presence of Ca2+. The activity of severin kinase itself was
not affected under these conditions because its autophosphorylation and
also phosphorylation of DS211C remained nearly unaltered in the
presence of Ca2+ (Fig. 4).
The addition of Ca2+ triggers a conformational change in
severin (28) that might render either the target amino acid or the
complete protein inaccessible for severin kinase. Since severin kinase
accepted also Mn2+-ATP as phosphate donor we tested the
Ca2+ dependence of the phosphorylation reaction under these
conditions as well. A similar Ca2+ dependence of severin
phosphorylation was found with Mn2+-ATP,
autophosphorylation of severin kinase, and phosphorylation of DS211C,
however, overall phosphorylation was not as pronounced as with
Mg2+-ATP (Fig. 4).

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Fig. 4.
Regulation of severin phosphorylation at the
substrate level. A, phosphorylation of severin by severin
kinase in the presence of Mg-ATP (Mg2+) or Mn-ATP
(Mn2+) as phosphate donor. The phosphorylation reactions
were carried out in the absence ( ) or presence
(Ca2+) of Ca2+. B,
phosphorylation of DS211C as a substrate. The positions of severin,
DS211C and the 62-kDa subunit of severin kinase are indicated. The
reactions were separated by SDS-PAGE in 12% gels, the gels stained,
dried, and autoradiographed.
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The 62-kDa Subunit of Severin Kinase Is a Member of the PAK Family
of Protein Kinases--
We used degenerate primers deduced from
tryptic peptide sequences of the 62-kDa protein to clone a 250-base
pair genomic fragment, and after screening two different cDNA
libraries we isolated two overlapping clones which represented the
full-length cDNA. The complete cDNA has a size of 1.54 kb and
encodes a polypeptide of 478 amino acids with a total molecular mass of
52,615 Da. The nucleotides preceding the start codon are characteristic
of Dictyostelium genes and there are two in-frame stop
codons upstream of it, indicating that the cloned cDNA harbors the
complete coding region. Northern blot analysis of growth phase
Dictyostelium cells showed one mRNA band with a size of
approximately 1.7 kb (data not shown). The difference between the
apparent molecular mass of 62 kDa in SDS-PAGE and the calculated
molecular mass of approximately 53 kDa could be explained by reduced
mobility of the polypeptide in SDS-PAGE. Similar differences in
apparent and calculated molecular mass were reported for the two
related kinases Krs-1 and Krs-2 (see below). Krs-1 and 2 have an
estimated molecular mass of 63 and 61 kDa on SDS-polyacrylamide gels
while the predicted molecular mass is 56.3 kDa for Krs-1 and 55.6 kDa
for Krs-2 (40). In this report the authors suggest that a highly acidic
region in the C-terminal domain could be responsible for the slightly
aberrant migration behavior in SDS-PAGE. The calculated pI of the
kinase is 6.7; all microsequences collected from the protein were
present in the cDNA deduced amino acid sequence.
The predicted protein sequence indicates a two-domain organization of
the protein (Fig. 5). The N-terminal part
with 276 residues constitutes the catalytic domain characteristic of
Ser/Thr- and Tyr-protein kinases. All 11 subdomains typically found in
these protein kinases (41) are present. The C-terminal domain
encompasses 202 residues and is rich in glutamine, threonine, and
proline residues. Most obvious are two glutamine-rich stretches between aa 323 and 347 and one threonine-rich stretch between aa 359 and 369 (9 out of 11 residues). Several proline residues (17 in total) are
scattered throughout the central part of the C-terminal domain between
residues 312 and 429. These regions could constitute binding interfaces
for regulatory proteins. In addition, a highly acidic region is present
between aa 290 and 306 with 10 negatively charged amino acids out of
17. Short acidic regions of unknown function have also been found
in other proteins from the PAK family including mammalian PAKs, Ste20p,
Krs-1 and 2, and SOK-1 (21, 40).

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Fig. 5.
Deduced amino acid sequence of the kinase
subunit of severin kinase in standard single-letter code. The
sequence is composed of two domains, an N-terminal kinase domain (aa
8-276) and a C-terminal domain of predicted regulatory function (aa
277-478, shaded). The 11 subdomains which are typical for
Ser/Thr and Tyr kinases are indicated by roman numerals. In
subdomain VIII of the catalytic domain, the PAK signature sequence is
shaded. Thin lines below the sequence mark N-terminal amino
acid sequences obtained by microsequencing of proteolytic peptides from
the 62-kDa protein.
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In data base searches with the program BLAST (38) the highest degree of
sequence similarity was observed in members of the PAK family of
protein kinases. These kinases share a highly conserved catalytic
domain and have the so-called PAK signature "GTPY/FWMAPE" in common
(Fig. 5). They can be subdivided into two groups based on their
structure and regulation. Ste20p, PAK1, MIHCK (42), and related PAKs
have a C-terminal kinase domain and a p21 binding motif in the
N-terminal part. In the GCK branch of the PAK family the catalytic
domain is positioned at the extreme N terminus (21). Based on its
primary structure and sequence homology, the 62-kDa subunit of severin
kinase clearly belongs to the GCK subfamily (Fig.
6A). Sequence comparisons of
its catalytic domain with the catalytic domains of the other kinases
revealed that the kinase subunit of severin kinase is most closely
related to human SOK-1 (75% identity) and the open reading frame
T19A5.2 from Caenorhabditis elegans (72% identity).
However, even with the most distant member in this comparison, Ste20p
from S. cerevisiae, the kinase from Dictyostelium
shared 42% sequence identity in the catalytic domain.

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Fig. 6.
Topology of the severin kinase subunit in a
schematic comparison (A) and position in an evolutionary
tree (B) of PAK family members. A, the
structures of GCK subfamily members (7 examples at the top) and true
PAKs (3 examples at the bottom) are schematically depicted. The kinases
were aligned according to the position of their catalytic domains
(white boxes). GCK subfamily members have a C-terminal
regulatory domain (dark gray boxes) and true PAKs an
N-terminal regulatory domain (light gray boxes) that
contains a p21-binding domain consensus sequence (black
boxes). The sequence identities (% identity) of the catalytic
domains are calculated relative to the 62-kDa subunit of severin
kinase. B, evolutionary tree of PAK family members. A
multiple sequence alignment of the catalytic domains of PAK family
members was calculated with the program Clustal of the UWGCG package.
The alignment was used to construct a phylogenetic tree with the
programs Protdist and Kitch of PHYLIP (Phylogeny Inference Package),
version 3.5c, by Joseph Felsenstein from the University of Washington.
True PAKs (dark gray box at the top) and GCK
subfamily members (lower boxes) split early in evolution.
Further separation among GCK members led to additional distinct
branches. The kinase subunit of severin kinase (arrow)
appears in a distinct branch together with human SOK1 and T19A5.2 from
C. elegans (dark gray box at the
bottom). The abbreviations used are: Dd, D. discoideum; Hs, Homo sapiens; Ce,
C. elegans; Mm, Mus musculus;
Sc, S. cerevisiae; Rn, Rattus
norvegicus. The sequences used have the following accession
numbers: PAK1 (U51120); PAK (P35465); PAK3 (U39738); STE20 (M94719);
CLA4 (X82499); MIHCK (U67716); KHS1 (U77129); Rab8IP (U50595); HPK1
(U66464); NIK (U88984); MST2 (U26424); Mess1 (U28726); MST1 (U18297);
T19A5.2 (U53153); SOK1 (U28726); NRK1 (D29980); SPS1 (A55162).
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To further clarify the relationship between PAK family members and the
kinase subunit of severin kinase, we calculated multiple sequence
alignments of the catalytic domains of PAK family members. In the
evolutionary tree derived from these alignments the members of the two
PAK branches are separated as expected, and for GCK subfamily members
the tree is split into two main branches, one formed by KHS1, Rab8ip,
HPK1, NIK, the other one by MST1, MST2, MESS1, NRK1, T19A5.2, SOK-1,
and the kinase subunit of severin kinase. SOK-1, T19A5.2, and the
kinase subunit of severin kinase are most closely related and listed
together (Fig. 6B).
A sequence alignment with human SOK-1 is shown in Fig.
7. The two proteins are 75% identical
and 84% similar in their catalytic domains. In addition, they share
significant sequence similarity in the C-terminal domain. Two long
regions of 44 and 58 amino acids with approximately 31% identity and
40% similarity, respectively, are present in this domain; the first
one is adjacent to the catalytic domain and the second one is located
at the extreme C terminus. A third short stretch of 16 amino acids in
the central part of the C-terminal domains displays 33% sequence
similarity and is flanked in the 62-kDa subunit of severin kinase by
two insertions of 22 (amino acid 320-341) and 51 (amino acid 358-408)
residues. Interestingly, when we compared the entire C-terminal domains of the Dictyostelium kinase and Rab8ip from mouse (43) or
GCK from human (44) with the program Bestfit we found only one short homologous region of 19 residues with about 37% sequence similarity. All 16 residues of the central C-terminal homology region of the Dictyostelium kinase and SOK-1 were contained in these 19 residues, thus raising the possibility for this sequence to constitute
an as yet unknown p21-binding motif.

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Fig. 7.
Sequence alignment of the kinase subunit of
severin kinase (62 kDa) and human SOK-1 (SOK-1). The sequence
alignment was done with the program Clustal from the UWGCG program
package. Identical and similar residues are indicated by a
star or a point, respectively. The
arrowhead marks the start of the regulatory regions. Both
sequences are closer related in their catalytic domains than in their
regulatory regions. A small stretch of similar amino acids (341-359 in
62 kDa) is also present in GCK and Rab8ip, and might be a putative
binding site for small GTPases. Residue numbers of both proteins are
shown on the right.
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DISCUSSION |
Based on in vitro phosphorylation assays we have
partially purified a severin kinase from cytoplasmic extracts of
D. discoideum. Severin kinase has a molecular mass of about
300 kDa and harbors a 62-kDa subunit that is closely related to
p21-activated protein kinases. Sequence comparisons of the catalytic
domains of selected PAKs and the severin kinase subunit clearly
identified the kinase as a new member of the GCK subfamily. It displays
the highest similarity to human SOK-1 that is activated by oxidant
stress (45). Both proteins are 75% identical in their catalytic and 31% identical in their regulatory domains and could therefore fulfill
a similar or even identical in vivo function.
Severin kinase is the first example of a GCK subfamily kinase with a
cytoskeletal protein as a possible in vivo target. In contrast to GCK subfamily members, several true PAKs have recently been
implicated in cytoskeletal reorganization or the regulation of
cytoskeletal proteins. Ste20p was found to bind to Bem1p which associates with actin (46) and PAK1 is thought to regulate actin organization in mammalian cells via an as yet unknown effector (47,
48). MIHCK from Dictyostelium and its homologue from Acanthamoeba phosphorylate the heavy chain of some of the
myosin I isozymes on a single serine or threonine residue and thereby stimulate their actin-activated Mg-ATPase activity 30-50-fold (49,
50). Cloning of the corresponding genes revealed that MIHCK is a member
of the PAK family and closely related to mammalian PAK and yeast Ste20p
molecules (42, 51). In gel overlay assays and affinity chromatography
experiments, MIHCK from Dictyostelium interacted with
GTP
S-labeled Rac1 and Cdc42, which probably bind to a conserved
p21-binding domain commonly found in the N-terminal regulatory domain
of true PAKs. Interestingly, in the presence of active Rac1 and Cdc42,
autophosphorylation of MIHCK increased from 1 up to 9 mol of phosphate
per mol of kinase concomitant with an approximately 10-fold stimulation
of the rate of myosin ID phosphorylation. These results suggest that
MIHCK directly links Cdc42/Rac signaling pathways to motile processes
driven by myosin I molecules (42).
For members of the GCK subfamily the putative regulatory role of the
C-terminal non-catalytic domain is not clear. In the case of MST1,
MST2, and SOK-1 it apparently has an inhibitory function because its
removal resulted in an increase in kinase activity (45, 52).
Furthermore, it has been shown that the C-terminal domains of MST1 and
MST2 mediate homo- and heterodimerization (52). Rab8ip, the murine
homologue of human GCK has been isolated in a two-hybrid screen as a
Rab8 interacting protein (43). This finding was surprising because
members of the GCK subfamily lack the conserved p21-binding domain of
16 amino acids found in the N-terminal regulatory domains of true PAKs
(21, 23). Thus it is possible that also other GCK subfamily members may
be regulated by small GTPases as well, but that a common binding motif
is not identified yet. We compared the sequences of the non-catalytic domain of GCK, Rab8ip, and the kinase subunit of severin kinase and
found a stretch of 19 similar amino acids (amino acids 341-359 in the
62-kDa subunit of severin kinase), that could constitute a binding site
for a small GTPase.
Like MIHCK and other kinases of the PAK family, severin kinase showed
strong and possibly multiple autophosphorylation (Fig. 2A)
which resulted in a severalfold activation of kinase activity (Fig.
3B). In addition, severin phosphorylation seemed to be
regulated at the substrate level since Ca2+ strongly
reduced phosphorylation of severin, whereas autophosphorylation of the
kinase and phosphorylation of DS211C were nearly unchanged (Fig. 4). In
two-dimensional gel electrophoresis, purified severin resolved in three
bands suggesting that also in vivo severin is subject to
phosphorylation. Treatment of purified severin with severin kinase
resulted in an additional more acidic spot (data not shown). It is at
present not clear whether phosphorylation influences one or more of the
in vitro activities of severin. This important issue is
difficult to resolve because one has to be able to obtain not only
fully phosphorylated severin, but also distinguish and characterize the
phosphorylation sites in native severin as opposed to recombinant
domains 1 (DS151) and domains 2 + 3 (DS211C). Phosphorylation of
fragmin, the Physarum homologue of severin, by a casein
kinase II enzyme had no effect on the in vitro activity of
fragmin (14). The authors speculate that phosphorylation of fragmin
could be associated with an intracellular redistribution similar to
gCAP39 which was shown to be preferentially associated with nuclear
preparations in the phosphorylated state (13).
Several members of the GCK subfamily are responsive to cellular stress.
Sps1p has been shown to become activated in response to nutrient
deprivation (53). Human Krs-1 and Krs-2, which are identical with MST1
and MST2, are activated upon treatment of cells with staurosporine,
okadaic acid, high concentrations of sodium arsenite, and extreme heat
shock at 55 °C (40, 54, 55). The activity of another member, human
SOK-1, was shown to be induced severalfold by oxidant stress, but not
by growth factors, alkylating agents, cytokines, heat shock, and
osmotic stress. It most likely controls a novel stress response pathway since it is not involved in already defined MAPK cascades (45). This
leads to the assumption that members from the GCK subfamily are
important for the response of eukaryotic cells to environmental stresses. The in vivo regulation of severin kinase is not
yet known. However, its close similarity to human SOK-1 and other kinases of the GCK subfamily suggests that it might also be activated in response to cellular stress. Possibly its activation leads to
phosphorylation of severin and connects an extracellular signal to the
cytoskeleton via an as yet unknown regulatory cascade. Disruption of
the severin kinase gene and in vivo labeling experiments should help to unravel the in vivo role of
Dictyostelium severin kinase.
We thank Dr. R. Kessin for sharing cDNA
libraries, Drs. Ch. Andréoli, R. Gräf, J. Faix, and A. A. Noegel as well as K.-P. Janssen and H. Felgner for stimulating
discussions and help during this work, and D. Rieger and S. Thiel for
excellent technical assistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF059534.