The high degree of conservation among the guanine nucleotide binding domains of septins raises the possi-bility that nucleotide binding and hydrolysis properties may be important for sep
Trang 1Yi-Wei Huang1, Mark C Surka1,3, Denis Reynaud2, Cecil Pace-Asciak2and William S Trimble1,3
1 Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada
2 Program in Integrative Biology, Hospital for Sick Children, Toronto, ON, Canada
3 Department of Biochemistry, University of Toronto, ON, Canada
Septins are a family of highly conserved guanine
nuc-leotide binding proteins that can assemble into
fila-ments and have been implicated in cytokinesis, cellular
morphogenesis, neuronal polarity, vesicle trafficking
and apoptosis in a wide range of organisms [1–7] First
identified in Saccharomyces cerevisiae, septins Cdc3p,
Cdc10p, Cdc11p and Cdc12p localize to the mother–
bud junction where they play important roles in bud
emergence and cytokinesis [8,9] At the bud neck their
appearance coincides with the presence of 10 nm
fila-ments that lie adjacent to the membrane in a concentric
pattern [8,9] In mammals, at least 13 septin isoforms
are predicted from genomic analysis [10], named
SEPT1-SEPT13 in humans [11], but for most of them
their biological functions remain unclear Septins can
be found in cytosolic fractions as heteromeric
com-plexes that appear to be comprised of most or all of the
septins expressed in a specific cell type [12] In inter-phase mammalian cells, most septins are organized into filamentous structures that often colocalize with actin bundles [13,14], or in some cases with microtubules [15,16], implying that septin filaments may be an important cytoskeleton component Immuno-isolated septin complexes from Drosophila [17] and yeast [18] have been shown to polymerize into filaments under favorable conditions In addition, recombinant com-plexes of SEPT2, SEPT6 and SEPT7 can be co-purified
in a 1 : 1 : 1 stoichiometry [12,19] These complexes can also polymerize into long filaments in vitro, indica-ting the capacity of septins for self-directed polymerization
All septins possess a highly conserved central core domain that has the potential to bind guanine nucleo-tide and a variable length amino terminal domain
Keywords
casein kinase II; GTP; GTPase kinetics;
phosphorylation; septins
Correspondence
W Trimble, Program in Cell Biology,
Hospital for Sick Children, 555 University
Avenue, Toronto, Ontario M5G 1X8, Canada
Fax: +1 416 8135028
Tel: +1 416 8136889
E-mail: wtrimble@sickkids.ca
(Received 24 February 2006, revised 8 May
2006, accepted 22 May 2006)
doi:10.1111/j.1742-4658.2006.05333.x
Septins are a family of conserved proteins that are essential for cytokinesis
in a wide range of organisms including fungi, Drosophila and mammals In budding yeast, where they were first discovered, they are thought to form a filamentous ring at the bridge between the mother and bud cells What regulates the assembly and function of septins, however, has remained obscure All septins share a highly conserved domain related to those found in small GTPases, and septins have been shown to bind and hydro-lyze GTP, although the properties of this domain and the relationship between polymerization and GTP binding⁄ hydrolysis is unclear Here we show that human septin 2 is phosphorylated in vivo at Ser218 by casein kinase II In addition, we show that recombinant septin 2 binds guanine nucleotides with a Kdof 0.28 lm for GTPcS and 1.75 lm for GDP It has
a slow exchange rate of 7· 10)5s)1for GTPcS and 5· 10)4s)1for GDP, and an apparent kcat value of 2.7· 10)4s)1, similar to those of the Ras superfamily of GTPases Interestingly, the nucleotide binding affinity appears to be altered by phosphorylation at Ser218 Finally, we show that
a single septin protein can form homotypic filaments in vitro, whether bound to GDP or GTP
Abbreviations
GTPcS, guanosine 5¢-[c-thio]triphosphate; Sf21 cells, Spodoptera frugiperda cells; TBB, tetrabromobenzotriazole.
Trang 2Most also possess a carboxyl-terminal predicted
coiled-coil domain The presence of characteristic motifs
within the GTPase region, including the P-loop (G-1
motif) as well as sequences resembling G-3 and G-4
motifs [20] has led to the classification of septins as a
novel group of GTPases [3] Many GTPases possessing
these motifs, such as members of the Ras superfamily,
function as molecular switches regulating many
essen-tial cellular processes by cycling between a GTP-bound
active state and a GDP-bound inactive state [20,21]
Due to their low intrinsic GTPase activity and slow
GDP-GTP exchange rates, ras family GTPases require
additional factors to promote GTPase activity and
nucleotide exchange in order to inactivate and activate
them, respectively Alternatively, for GTPases like
b-tubulin, the binding of GTP alters the conformation
of the protein to promote polymerization while
hydro-lysis of GTP serves as a timing mechanism to control
polymer turnover
The high degree of conservation among the guanine
nucleotide binding domains of septins raises the
possi-bility that nucleotide binding and hydrolysis properties
may be important for septin function, but their precise
role remains unclear Septin complexes isolated from
a variety of organisms and recombinant septin
com-plexes contain stoichiometric amounts of bound
guan-ine nucleotide with the majority being GDP [12,17,22]
Septins have been reported to bind guanine nucleotide
and hydrolyze GTP to GDP [13,17,23,24], but the
sig-nificance of this with respect to septin polymerization
has remained controversial Mendoza et al [23] have
reported that GTP binding induces Xenopus SEPT2
filament assembly in vitro, however, this
polymeriza-tion does not require GTP hydrolysis Likewise, yeast
septins unable to hydrolyze GTP could form septin
neck rings in vivo while mutants unable to bind GTP
did not form neck rings and could not polymerize into
filaments in vitro [25] Sheffield et al [24] reported that
GTP binding and hydrolysis may be important for
mammalian septin heterodimer formation In contrast,
Kinoshita et al [12] demonstrated that septin
com-plexes can self-assemble into filaments while
predomin-antly bound to GDP and in the absence of guanine
nucleotide hydrolysis Clearly, a thorough analysis of
the GTPase enzyme kinetics will be important in
gain-ing insights into the contribution of the GTPase
domain to septin function
In this study, we examined the GTPase properties of
mammalian SEPT2 We show that recombinant
SEPT2 produced via baculovirus expression has
meas-urable GTP binding and hydrolysis kinetics
compar-able to that of Ras superfamily GTPases The purified
protein has the capacity to polymerize into long
fila-ments when loaded with GTP or GDP Moreover, we show that the endogenous protein in HeLa cells, like that produced in insect cells, is phosphorylated by casein kinase II and that this phosphorylation alters nucleotide binding
Results
Septin 2 is phosphorylated by casein kinase II Recombinant Septin 2 (SEPT2) was produced by bacu-lovirus-mediated expression in Sf21 cells, and we previ-ously examined its mass by laser desorption⁄ ionization quadrupole⁄ time-of-flight tandem mass spectrometry and noted that the recombinant protein was singly phosphorylated Direct peptide mapping and sequence analyses on various enzymatic digests identified a novel phosphorylated site at residue Ser218 in vivo This unique monophosphorylation at Ser218 was confirmed
by site-directed SEPT2 mutagenesis of Ser218 to Ala,
as similar analysis of the mutated protein showed no evidence of phosphorylation [26] Residue S218 is detected by a variety of phosphorylation prediction software to be a high probability casein kinase II site The detection of SEPT2 phosphorylation in Sf21 cells raised the question of whether endogenous septins are phosphorylated in mammalian cells in vivo To begin to address this possibility HeLa cells were cul-tured in the presence of [32P]-orthophosphate for 5 h
to radiolabel phosphoproteins in vivo Cells were then solubilized in a lysis buffer containing 0.5% Triton X-100 and phosphatase inhibitors The cleared lysate was then subjected to immunoprecipitation using anti-body specific to SEPT2 We have previously shown [15] that immunoprecipitation of SEPT2 results in the co-immunoprecipitation of septins 6, 7 and 9 in HeLa cells As shown in Fig 1, several phosphoproteins were immunoprecipitated with anti-SEPT2 antibodies and the mobilities of these bands are consistent with each
of the septins that co-immunoprecipitate with SEPT2 from HeLa cells [15] The position of SEPT2 was con-firmed by western blotting (data not shown) and is indicated by an asterisk
As a means of identifying the possible kinases responsible for SEPT2 phosphorylation we performed
an in-gel kinase assay In this assay recombinant SEPT2, expressed as a GST-fusion protein in bacteria,
is incorporated into the polyacrylamide matrix prior to electrophoresis Lysates of HeLa cells were electro-phoresed through the gel, and then renatured to iden-tify the presence of any kinases capable of using SEPT2 as a substrate In this assay, we found two prominent bands between 40 and 50 kDa consistent
Trang 3with the mobilities of the a and a¢ subunits of casein
kinase II (Fig 2A) To determine if these were casein
kinase 2, recombinant casein kinase II was
electro-phoresed beside the cell lysate and gave a similar
banding pattern In addition, both the recombinant
casein kinase II and cell lysate bands were sensitive to
heparin, a casein kinase II inhibitor (Fig 2B) The
identity of casein kinase II as the responsible kinase
was further supported by transfection experiments in
which a myc-tagged SEPT2 construct was expressed in
HeLa cells in the presence of [32P]-orthophosphate
Cells were then incubated for 6 h with the casein
kin-ase inhibitor tetrabromobenzotriazole (TBB) at
differ-ing concentrations and then immunoprecipitated with
anti-myc IgG As seen in Fig 2C, myc-SEPT2
phos-phorylation was inhibited in a dose-dependent manner
by TBB
Prosite analysis of SEPT2 sequence revealed five
potential casein kinase II sites located at amino acids
84, 98, 120, 198 and 218 To determine if the site
phorylated by Sf21 cells [26] was the same one
phos-phorylated in HeLa cells we utilized site-directed mutagenesis to convert serine 218 to alanine Cells were then transfected with myc-SEPT2WT or myc-SEPT2S218A, or mock transfected, labeled with [32P]-orthophosphate, and immunoprecipitated with anti-myc As seen in Fig 2D, only myc-SEPT2WT, but not myc-SEPT2S218A, was phosphorylated in logarith-mically growing HeLa cells
Septin 2 binds and hydrolyses GTP Like all septins characterized to date, SEPT2 can bind and hydrolyze GTP in vitro, yet its kinetic properties are not well known To measure GTP binding and hydrolysis kinetics, human SEPT2 was expressed in Sf21 cells and purified from cell lysates with nickel-chelated affinity chromatography to more than 95% purity based on SDS⁄ PAGE (Fig 3) Identity of the protein was confirmed by immunoblot analysis using a polyclonal antibody raised against SEPT2 [14] We found that purification in 20% glycerol was necessary
to stabilize this protein As well, the addition of GDP
to the purification buffer system further stabilized the protein, leading to full nucleotide binding activity (see below)
We first sought to determine if baculovirus-expressed SEPT2 co-purifies with nucleotide To define the nuc-leotide bound state of the purified proteins we used reverse-phase HPLC and observed (Fig 4A) that
1 mole of SEPT2 bound approximately 1.3 moles of nucleotide, consistent with results reported for mam-malian and Drosophila septin complexes [12,17] The GTP to GDP ratio was about 0.1, lower than that observed for immuno-isolated Drosophila septins [17] This is significantly different from Xenopus laevis Sept2, which was found to be nucleotide free when purified from bacteria [23] We therefore examined the nucleo-tide status of SEPT2 when purified from Escherichia coli and found that 60% of the protein contained guanine nucleotides in a GDP–GTP ratio of 1.4 : 1 (data not shown) Figure 4B shows that the nucleotides are efficiently exchanged and can be removed from the protein (about 10% of the nucleotides remain bound)
At high Mg2+ concentrations GTPcS replaced GDP efficiently, but could be readily stripped from the pro-tein during wash steps when the propro-tein is bound on nickel beads in the low Mg2+concentrations due to its rapid GTPcS off-rate under these conditions
Using a filter-binding assay we measured both the equilibrium constant (Kd) and dissociation rate con-stant (koff) of guanine nucleotides for SEPT2 To mon-itor GTP binding, a nonhydrolysable GTP analog, GTPcS, was used Considering that Mg2+is known as
Fig 1 Septin 2 co-immunoprecipitates with several
phospho-proteins Immunoprecipitation with affinity purified anti-SEPT2
serum from cells labeled with [32P]-orthophosphate reveals that
SEPT2 and several co-precipitating proteins are phosphorylated in
vivo No radioactive bands were detected when the preimmune
serum was used (left lane) The asterisk denotes the band detected
by affinity purified anti-SEPT2 serum on western blots (not shown).
Trang 4an essential cofactor for many GTP-binding protein
functions [27], we examined whether septin nucleotide
binding and hydrolysis were affected by Mg2+
concen-tration Figure 5A,B represents the binding results of a
set of independent experiments with different Mg2+
concentrations for SEPT2 While binding of GTPcS
was saturable at all Mg2+ concentrations, it was
clearly enhanced by Mg2+ levels in the physiological
range (0.5–5 mm) (Table 1) The wild-type protein
showed a binding stoichiometry of 1, indicating that 1
molecule of protein bound 1 molecule of nucleotide
for both GTPcS and GDP in each of the Mg2+
con-centrations tested The data revealed a hyperbolic
curve that was best fit to a single bimolecular binding
model Scatchard analysis is shown in the inset and the
Kd for GTPcS was measured to be 0.28 ± 0.06 lm in
5 mm MgCl2 and 3.37 ± 1.42 lm in 0.01 mm MgCl2
Thus, the approximate 12-fold difference between low and high Mg2+ concentrations indicates the import-ance of Mg2+ in GTP-binding Interestingly, this
Mg2+dependence was not observed for GDP binding (Fig 5B and Table 1) The Kdvalues were very similar
in the low micromolar range at different Mg2+ con-centrations Interestingly, when the nonphosphorylat-able S218A form of SEPT2 was examined, it had a much higher Kd value of 2.5 lm for GTPcS and 4.4 lm for GDP (Table 1) This was very similar to the values obtained for SEPT2 produced in E coli (1.7 lm for GTPcS and 6.4 lm for GDP) (data not shown), suggesting that serine phosphorylation by casein kinase II decreases the affinity of SEPT2 for guanine nucleotides
We next examined the effect of Mg2+ on the dis-sociation rate of GTPcS and GDP from SEPT2
C
D
Fig 2 SEPT2 is phosphorylated at S218 by casein kinase II (A) In-gel kinase assay reveals that Sept )2 is an efficient substrate for kinases that have a mobility between 40 and 50 kDa Gels were polymerized with GST (left lanes) or GST-SEPT2 (right lanes) and duplicate samples
of HeLa cell lysates were electrophoresed The gels were renatured in the presence of [ 32 P]-ATP as described in the methods and then autoradiographed (B) In-gel kinase assays show that commercial casein kinase II (left panel, right lane) gave bands comparable to the cell ly-sate (left panel, left lane) and both bands were inhibited by heparin (right panel) (C) Increasing concentrations of TBB reduced SEPT2 phos-phorylation Cells were transiently transfected with myc-SEPT2, then immunoprecipitated, labeled with [ 32 P]-orthophosphate, and then lysed and lysates immunoprecipitated with anti-myc IgG Increasing amounts of TBB caused a reduction in the labeling of myc-SEPT2 (D) Cells were either mock transfected or transfected with myc-SEPT2, labeled with [ 32 P]-orthophosphate, and then immunoprecipitated with anti-myc as above Autoradiography reveals that the wild-type protein is significantly labeled but SEPT2 S218A is not (top panel) A western blot of lysates was probed with anti-SEPT2 to reveal that both constructs were equivalently expressed (lower panel, upper band), and at levels not greatly above endogenous SEPT2 levels (lower panel, lower band).
Trang 5Figure 6A,B represents a set of independent experi-ments for the dissociation of GTPcS and GDP, respect-ively, from SEPT2 with different Mg2+concentrations
Fig 4 Reverse-phase HPLC monitoring of guanine nucleotides
bound on SEPT2 Purified SEPT2 (240 pmol; A) was extracted as
described in the methods and bound nucleotides were fractionated
by HPLC on a Nova-Pack C18 column (B) A three molar ratio of
GTPcS was added to the purified protein with 5 m M MgCl2 and
incubated at room temperature for 3 h to exchange GDP with
GTPcS The protein was then reloaded onto a nickel column to
remove unbound nucleotides Bound GTPcS quickly released and
washed away during wash steps with buffer A without Mg 2+ and
nucleotide The protein was then eluted in 150 m M imidazole.
(B) shows only about 10% of the nucleotides still bound to the
pro-tein (C) Control reveals the HPLC profile of a mixture of 240 pmol
of GDP, GTP and GTPcS Absorbance at 254 nm is indicated in
arbitrary units.
Fig 3 Purification results of His6-tagged SEPT2 wild-type
over-expressed in Sf21 cells (A) Purification of SEPT2 A Commassie
brilliant blue stained 12% SDS ⁄ PAGE gel reveals the purification of
SEPT2 N-terminal His6-tagged SEPT2 proteins were overproduced
in Sf21 cells, accounting for about 10–15% of the total protein in
the insect cell lysate (L) Samples were sedimented at 110 000 g
and the majority of the SEPT2 protein remained in the supernatant
(S) After passage over Ni-NTA columns the flow-through (FT) was
depleted of SEPT2, which mainly remained bound to the column
during the washes (W1, W2) and eluted (E) as a single species in
150 m M imidazole.
A
B
Fig 5 Guanine nucleotide binding of SEPT2 (A) Equilibrium binding curves of GTPcS with different concentrations of Mg2+ Data were plotted by fitting to a bimolecular binding equation Scatchard analy-sis is shown in the inset with the ratio of the concentration of bound GTPcS over the concentration of SEPT2 divided by the free GTPcS ([b] ⁄ [SEPT2] ⁄ [f] – y axis) plotted against the ratio of bound GTPcS over the SEPT2 concentration ([b] ⁄ [SEPT2] – x axis) (B) Equilibrium binding curves of GDP with different concentration
of Mg 2+ Data were plotted by fitting to a bimolecular binding equa-tion Scatchard analysis is shown in the inset with the ratio of the concentration of bound GDP over the concentration of SEPT2 divi-ded by the free GDP ([b] ⁄ [SEPT2] ⁄ [f] – y axis) plotted against the ratio of bound GDP over the SEPT2 concentration ([b] ⁄ [SEPT2] –
x axis).
Trang 6From these panels we can see the same phenomenon as
seen for the Kd measurements, that at different Mg2+
concentrations the dissociation of GTPcS is much more
greatly affected than that of GDP dissociation Surpris-ingly, the data best fit to either single or bi-exponential decay models depending on the Mg2+ concentration The summary of the koff rates of SEPT2 for GTPcS and GDP fit to a single exponential decay model is pre-sented in Table 2 The dissociation half-life for GTPcS
in low Mg2+is 1.24 min and in high Mg2+is 165 min The dissociation half-life for GDP in low Mg2+ is 3.62 min and that in high Mg2+ is 23.1 min Again, these results show Mg2+dependence for GTPcS disso-ciation, which has > 130-fold difference between low and high Mg2+concentrations while that for GDP has only a six-fold difference In similar experiments carried out with the S218A mutant, we found that the kofffor GTPcS and GDP at high Mg2+ are not significantly different from those of SEPT2WT (Table 2) with the dissociation half-life 182 min and 16.5 min for GTPcS and GDP, respectively, at high Mg2+ concentration Since the Kd for GTPcS of the mutant is significantly increased, it implies that the association rate constant (kon) decreases
In order to measure kon and hydrolysis rate con-stants, it is first necessary to produce apoprotein devoid of nucleotide, but all attempts to remove nuc-leotide from the protein resulted in protein instability and insolubility Hence, it was not possible to measure
konand hydrolysis rate constants by these means
As we were unable to measure hydrolysis rate con-stants directly, we used radioactive assays to measure
Table 1 Summary of the GTPcS and GDP dissociation constants and stoichiometrical binding site (n) for wild-type SEPT2 and S218A mutant The data for SEPT2 were best fit with a single binding model while n represents the number of binding sites per protein molecule The values represent mean ± SD of at least three independent experiments ND, not done.
MgCl 2 (m M )
5 0.28 ± 0.06 0.98 ± 0.11 2.46 ± 0.6 0.73 ± 0.1 1.72 ± 0.15 0.94 ± 0.28 4.40 ± 1.8 0.50 ± 0.1
A
B
Fig 6 Guanine nucleotide dissociation from SEPT2 (A) GTPcS
dissociation curves with different concentration of Mg 2+ (B) GDP
dissociation curves with different concentrations of Mg2+
Radioiso-topes used for GTPcS and GDP binding were 35 S-GTPcS and
3 H-GDP, respectively Data were plotted by fitting to single or
bi-exponential decay equations.
Table 2 Summary of the GTPcS and GDP dissociation rate con-stants for SEPT2 The data were fit to a single exponential decay model The values represent mean ± SD of at least three independ-ent experimindepend-ents ND, not done.
MgCl2 (m M )
GTPcS k off ( · 10)3s)1) GDP k off ( · 10)3s)1)
5 0.07 ± 0.02 0.06 ± 0.02 0.5 ± 0.1 0.7 ± 0.2
Trang 7SEPT2 steady-state kinetic constants, kcat and Km at
different Mg2+ concentrations In Fig 7, we show a
representative experiment of SEPT2 at 5 mm Mg2+,
revealing a hyperbolic initial velocity curve that is a
function of GTP concentration, and fits well with the
Michaelis–Menten equation A Lineweaver–Burke plot
of the data is shown in the inset The apparent kinetic
constant for SEPT2 is summarized in Table 3 The kcat
and Km values are not significantly different at 5 and
0.5 mm MgCl2; however, GTPase activity cannot be
clearly measured without Mg2+ in the buffer system
The kcatvalue for the SEPT2S218A mutant is similar to
that of the SEPT2WT However, consistent with the Kd
value, Kmis also four-fold larger than that of the wild-type
Previous studies had reported the ability of septins
to form filaments in vitro [12,17,18,24] We therefore set out to determine if baculovirus-expressed SEPT2 could form homo-oligomeric filaments in vitro Consis-tent with previously published results, we observed fila-ments of a variety of lengths similar to those seen for Xenopus SEPT2 [23] and for Drosophila and yeast septin complexes [17,18] SEPT2 filaments were detect-able regardless of which nucleotide was present (GTP
or GDP) and typically these filaments were more than
5 lm in length and appeared to be bundles of fila-ments of approximately 20–40 nm in diameter con-taining several intertwined filaments in the bundles (Fig 8) However, we did note differences in the rate
of filament formation Filaments formed within 30 min when protein was loaded with GTPcS or GTP (not shown), but took up to 6 h to achieve detectable fila-ments when loaded with GDP (Fig 8B)
Discussion
In this paper, we describe the first detailed kinetic study of the GTP binding and hydrolysis properties of
a single septin protein and demonstrate that phos-phorylation of serine 218 by casein kinase II alters these properties Previous studies have focused on sep-tin complexes because single sepsep-tin proteins expressed
in bacteria were found to be unstable and have severely altered nucleotide binding [24,28] precluding their analysis Some information has been gained by the study of septin complexes, but mammalian septin complexes expressed in E coli had extremely slow nuc-leotide exchange rates [24] This is similar to the poor exchange rates seen for endogenous septin complexes immunoisolated from Drosophila [17] and yeast [22] Complexes comprised of different septin isoforms would reflect the binding and hydrolysis properties of each protein Indeed, it has been reported that yeast septins Cdc10 and Cdc12 contributed the majority of
Fig 7 GTP hydrolysis kinetics of SEPT2 SEPT2 GTP hydrolysis
kinetic constants were measured in a mixture with 40 m M Tris,
pH 7.5, 10% glycerol, 0.5 mgÆmL)1 bovine serum albumin, 5 m M
MgCl 2 , 1 m M EDTA, 5 m M dithiothreitol, a fixed concentration of
purified protein and varying concentrations of 32 P-GTP at room
tem-perature Data from a representative experiment show the
hyperbo-lic initial velocity curve as a function of GTP concentration and
were plotted by fitting with the Michaelis–Menten equation The
Lineweaver–Burke plot of the data is shown in the inset Reaction
conditions for all panels are specified in experimental procedures.
Figures are representative of the results of several independent
experiments.
Table 3 Kinetic constants of GTP hydrolysis of Sept2 wild-type and mutant S218A The Kmand kcatvalues represent mean ± SD of at least three independent experiments The kcats values were calculated from the Vmaxvalues with the molecular mass of 43.5 kDa NM, not meas-urable; ND, not done.
MgCl2(m M )
kcat (x10)4s)1)
Km (l M )
kcat⁄ K m ( · 10)4s)1Æl M )1)
kcat (x10)4s)1)
Km (l M )
kcat⁄ K m ( · 10)4s)1Æl M )1)
Trang 8the GTP binding to the yeast septin complex [25].
Also, complexes of mammalian septins comprised of
different septin isoforms also exchanged guanine
nucle-otides at different rates [24] We therefore investigated
the possibility that insect cell expression may provide
protein more suitable for these types of analyses
Moreover, we have recently shown that SEPT2
expressed in Sf21 cells is phosphorylated at a single
site [26], raising the possibility that the expression
system may affect its properties We now show that
baculovirus expression of SEPT2 unable to be
phos-phorylated at this residue, as well as SEPT2 expressed
in bacteria and therefore not phosphorylated, had
sig-nificantly different kinetic properties By examining a
single septin we have eliminated the complicating
dif-ferences in kinetics that likely exist between septin
iso-forms when one examines septin complexes
Recombinant SEPT2 expressed in baculovirus was
stable for several months in)80 C without significant
loss of binding activity (data not shown) However,
when the nucleotide was removed from SEPT2, the
protein quickly became unstable and lost nucleotide
binding and filament-forming properties This
phenom-enon suggests that nucleotide binding is important in
maintaining proper protein conformation This is also
consistent with what has been seen in many nucleotide
binding proteins For example, removing the tightly
bound nucleotide from p21H–ras renders the protein thermally unstable [29], although apoproteins can be produced for Ras under specific conditions [30] Con-centrated nucleotide-free Ran, a Ras-related nuclear, immediately precipitates on diluting to working con-centration (1–2 lm) [31] Similarly, addition of GDP during the purification is critical in obtaining fully act-ive Dictyostelium elongation factor 1A [32] while nuc-leotide-free actin denatures rapidly at a rate of 0.2 s)1 [33]
The GTPcS Kd of SEPT2 is 0.28 lm and that of GDP is 1.7 lm in the presence of physiological Mg2+ concentrations These values are about an order of magnitude lower than those observed for complexes
of yeast septins expressed in bacteria Complexes of Cdc12p, Cdc11p-Cdc12p and Cdc3p-Cdc10p-Cdc12p had Kd values of 1.6, 6.2 and 5.9 for GTPcS However, they are much more reminiscent of the Kd values for GTPcS obtained for SEPT2 S218A (2.5 lm) or for SEPT2 when expressed in E coli (1.7 lm) It would be of interest to determine if post-translational modifications alter the kinetic properties
of yeast septins These binding constants are signifi-cantly different from those of the Ras family members (from picomolar to nanomolar) but similar to those
of the Rho family of small GTPases [34,35] Interest-ingly, we observed a Mg2+-dependent phenomenon
Fig 8 SEPT2 forms homotypic filaments in
GTP and GDP bound states Negative stain
electron micrographs of SEPT2 filaments
found after loading the protein with GTP (A),
GDP (B) or GTPcS (C), followed by 6 h of
incubation Scale bars on these figures
rep-resent 500 nm Higher magnification of
GTPcS-bound filaments (D) reveals lateral
bundles of SEPT2 protein Scale bar
repre-sents 100 nm.
Trang 9for GTPcS binding while GDP binding was Mg2+
independent Also, we made the surprising observation
that the decay fitting models best fit either single- or
bi-exponential curves depending on Mg2+ levels This
likely reflects different degrees of polymerization of the
protein that occur during the assay Unfortunately, we
were unable to distinguish the properties of
filamen-tous septin complexes and monomeric septins since
SEPT2 spontaneously polymerized during the course
of the assays
When comparing the off-rates, the value measured
for SEPT2 (t½¼ 165 min) appears to be quite similar
to that found for a SEPT2⁄ 6 ⁄ 7 complex expressed in
bacteria (t½¼ 150 min) [24] This nucleotide binding
property is not fully consistent with either Ras or Rho
proteins For example, p21H–ras shows Mg2+
-depend-ent guanine nucleotide binding behavior with a
>500-fold difference in GDP koffin the presence and absence
of Mg2+ while Mg2+ has no significant influence on
Kon [30,36,37] In the case of Rho family proteins,
RhoA, Cdc42 and Rac1 show similar Kd values for
GTPcS and GDP binding in the presence or absence
of Mg2+ although in the absence of Mg2+ their off
rates significantly increased, indicating that their
nuc-leotide association rates increase in parallel and the
intrinsic catalytic activities are not significantly affected
by Mg2+ [34,35] Unfortunately, due to our inability
to produce SEPT2 in the nucleotide-free state, we were
unable to measure kon and hydrolysis rate
con-stants The apparent kcatfor SEPT2 as determined by
steady state kinetics is very low (2.7· 10)4s)1 or
0.016 min)1), remarkably similar to steady state values
measured from yeast Cdc3p⁄ Cdc12p binary complexes
0.019 min)1 [28] In this case, it is thought that, as
Cdc3p does not exchange GTP, this value entirely
derives from Cdc12p-mediated hydrolysis These values
are more than an order of magnitude higher than the
value measured for singly expressed Cdc10p and
Cdc12p [25] This Kcat is similar to the intrinsic
GTPase rate of p21ras proteins (3.4–5 · 10)4s)1)
[20,38] and that of Rho family members RhoA, RhoB,
S cerevisiae Cdc42 and Caenorhabditis elegans Cdc42
(3.4 · 10)4s)1) [39] Like these proteins, SEPT2 does
not appear to have self-stimulatory GAP activity when
incubated at increasing concentrations (data not
shown) This is in contrast to several other Rho family
members such as RhoC, human Cdc42 and Rac2,
which contain a self-stimulatory GAP activity [39]
The kcat also depends on Mg2+ since it cannot be
clearly measured following depletion of Mg2+,
consis-tent with the fact that Mg2+ is an essential cofactor
for most GTPases The crystal structure of RhoA
revealed that elimination of the Mg2+ ion induced a
significant conformational change in the switch I region that opens up the nucleotide-binding site and suggested that a guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation for GDP⁄ GTP exchange [40] The
Mg2+-dependent and -independent binding properties
of SEPT2 for GTPcS and GDP, respectively, may also indicate different conformations in recognizing triphos-phate and diphostriphos-phate guanine nucleotides The simi-larity of the Kd, koff and kcatof septins and members
of the Rho family could be taken to imply that septins require GTP exchange and GTPase activating proteins
to complete the GTP binding and hydrolysis cycle effi-ciently However, at present it is not known how rap-idly these events would need to take place to support septin function Indeed, a recent report has suggested that, as is the case for a-tubulin, the role of guanine nucleotides in septins may be to ensure structural integrity of the protein [22]
The ability of mammalian SEPT2 to polymerize
in vitro, similar to that seen for Xenopus SEPT2 [23], indicates that the formation of septin filaments does not require an ordered array of a set of septins from distinct families, as has been recently postulated [41] Whether other mammalian septins also have this capa-city is not known, but it is interesting that immunopre-cipitation of septins from cells routinely results in the co-precipitation of other septins in near stoichiometric ratios [12,15], indicating that the formation of homo-polymers is not typical in vivo
It was also noteworthy that the S218A substitution near the C-terminus of the protein had a significant effect on the GTP binding property of SEPT2 This suggests that either the phosphorylation event results in changes in intraprotein conformation that affect the folding of the GTPase domain, or that inter-protein interactions between the C-terminal domain of one molecule of SEPT2 and the GTPase domain of another influence its properties We did not notice a difference
in the capacity of S218A mutant protein to polymerize into long filaments, although it did appear to be less efficient at forming thick bundles of filaments (data not shown) It will be of interest to determine if septin poly-merization is dynamically regulated in the cell, and if
so, to what extent phosphorylation status is involved
Experimental procedures
Expression and purification of recombinant SEPT2 and mutants
Human SEPT2 cDNA [14] (accession number T19030) was subcloned into the baculovirus expression vector
Trang 10pFast-BacHTb (Invitrogen, Burlington, ON, Canada) following
PCR amplification The SEPT2S218Amutant was generated
using the QuikChangeTM site-directed mutagenesis kit
(Stratagene, La Jolla, CA, USA) Subcloned and
mutagen-ized cDNA sequences were verified by dideoxynucleotide
sequencing Note that in She et al [26], the site of
phos-phorylation was indicated as Ser248 in the protein due to
the 30 amino acid tag added to its N-terminus During the
course of these studies, we noted that a PCR error had
resulted in a R331H substitution that was found in all
con-structs after the completion of most of the studies described
herein Given the conserved nature of this residue we
there-fore mutated the residue back to arginine and found that
none of the properties of the protein were altered by this
mutation (data not shown) This was not surprising, as a
large deletion including this region of the protein had
previ-ously been shown to have no effect on filament formation
or GTPase activity in X laevis Sept2 [23] The proteins
were overproduced in Sf21 insect cells as recommended by
the manufacturer Briefly, cells were harvested 48 h
postin-fection by centrifugation at 1000 g for 10 min, washed once
with phosphate-buffered saline (NaCl⁄ Pi), pelleted again
and kept at )80 C for later use The purification steps
were carried out at 4C unless otherwise specified Before
purification, cell pellets from about 6· 108
cells were sus-pended in 20–40 mL of buffer A (40 mm Tris, pH 8.0,
100 mm NaCl, 20% glycerol, 0.4 mm phenylmethylsulfonyl
fluoride, 1 lg mL)1 of each leupeptin and pepstatin A,
40 lm GDP and 8 mm imidazole) Cells were disrupted by
sonication and centrifuged at 110 000 g for 60 min The
supernatants were loaded onto Ni-NTA column (Qiagen,
Mississauga, ON, Canada) pre-equilibrated with the same
buffer, washed twice with buffer A without GDP in 10 mm
and 28 mm imidazole concentrations, respectively Proteins
were eluted with buffer A without GDP in 150 mm
imidaz-ole Proteins were routinely dialyzed in buffer D (40 mm
Tris, pH 7.5, 20 mm NaCl, 20% glycerol, 1 mm EDTA and
1 mm dithiothreitol) although no difference was observed
when 200 mm NaCl was added Protein concentration was
determined using the Bradford reagent (Bio-Rad,
Mississ-auga, ON, Canada) The dialyzed proteins were stored at a
concentration of1 mgÆmL)1at)80 C and they were
sta-ble for several months
In vivo [32P]-orthophosphate labeling and
immunoprecipitation
SEPT2WT or mutant SEPT2S218A were subcloned into a
modified pcDNA3.1 (Invitrogen) vector that contained myc
epitope at the N-terminus HeLa cells were transiently
transfected with myc-tagged SEPT2WTor SEPT2S218Ausing
Lipofectamine (Invitrogen) according to the manufacturer’s
instructions Eighteen hours post-transfection, cells were
washed twice with phosphate-free Dulbecco’s modified
Eagle’s medium Cells were then grown in the presence of
0.75 mCi [32P]-orthophosphate for 5 h at 37C Cells were lysed with lysis buffer (40 mm Tris, pH 7.5, 20% glycerol, 0.2 m NaCl, 2 mm EDTA, 0.5% Triton X-100, 1 lm oka-daic acid sodium salt, 50 mm NaF, 0.4 mm orthovanadate,
1 mm phenylmethylsulfonyl fluoride, 1 lgÆmL)1 of each of the protease inhibitors leupeptin and pepstatin A SEPT2 was immunoprecipitated with mouse antic-myc monoclonal antibody IgG1 (9E10, Santa Cruz biotechnology, Santa Cruz, CA, USA) The immunocomplexes were washed four times with wash buffer (lysis buffer with 0.1% TritonX-100 and without proteases inhibitors) Proteins were eluted with SDS⁄ PAGE sample buffer, separated by SDS ⁄ PAGE and blotted Labeled SEPT2 was visualized by autoradiography and quantitated using PhosphorImager
For endogenous SEPT2 immunoprecipitations, HeLa cells were grown in 10-cm dishes to 70% confluence before proceeding to label and collect cells as described above Affinity purified anti-SEPT2 serum (3 lg) was added
to 40 lL of 50% protein-A agarose beads (Invitrogen) and rotated for 1 h at 4C The beads were washed three times with HKA buffer, and the cell lysate was added along with NaCl to a final concentration of 150 mm The mixtures were rotated at 4C for 1–2 h and washed five times with ristocetin-induced platelet agglutination (RIPA) buffer (1% Nonident P-40, 1% sodium deoxycholate, 0.1% SDS,
150 mm NaCl, 0.01 m sodium phosphate pH 7.2, 2 mm EDTA) containing phosphatase inhibitors
Inhibition of CK2 in vivo HeLa cells transfected with myc-SEPT2WT for 18 h were first washed twice with phosphate-free Dulbecco’s modified Eagle’s medium and incubated with indicated concentra-tions of TBB (Calbiochem, EMD Biosciences, San Diego,
CA, USA), a specific CK2 inhibitor [42], for 1 h Cells were then grown in the presence of 0.75 mCi [32 P]-orthophos-phate together with the same amounts of TBB for 5 h at
37C and treated as described above
In-gel kinase assays Purified GST or GST-Septin2 was copolymerized in the sep-arating gel of a 10% SDS⁄ PAGE at a concentration of 0.1 mgÆmL)1 Triton solubilized HeLa cell lysates (30 lg) and⁄ or 10 units of purified CK2 enzyme (Promega, Madi-son, WI, USA) were loaded and electrophoresed SDS was removed from the gels by washing twice in buffer A (50 mm Tris⁄ HCl, pH 8.0 containing 20% 2-proponal) for 30 min, followed by washing in buffer B (50 mm Tris⁄ HCl, pH 8.0 containing 5 mm 2-mercaptoethanol) Proteins were dena-tured by washing gels in two changes of buffer B containing
6 m guanidine chloride for 60 min Renaturation was carried
at 4C for 30 min in buffer C (50 mm Tris ⁄ HCl, pH 8.0;
5 mm b-mercaptoethanol; 0.04% Tween-20) The gels were preincubated in kinase buffer (40 mm Hepes, pH 7.5,