Báo cáo khoa học: GTP binding and hydrolysis kinetics of human septin 2 pot

GTP binding and hydrolysis kinetics of human septin 2 Yi-Wei Huang 1 , Mark C. Surka 1,3 , Denis Reynaud 2 , Cecil Pace-Asciak 2 and William S. Trimble 1,3 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 K d of 0.28 lm for GTPcS and 1.75 lm for GDP. It has a slow exchange rate of 7 · 10 )5 s )1 for GTPcS and 5 · 10 )4 s )1 for GDP, and an apparent k cat value of 2.7 · 10 )4 s )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. 3248 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS Most 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 [ 32 P]-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 Y W. Huang et al. GTPase properties of human SEPT2 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3249 with 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 [ 32 P]-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 phos- 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-SEPT2 WT or myc- SEPT2 S218A , or mock transfected, labeled with [ 32 P]-orthophosphate, and immunoprecipitated with anti-myc. As seen in Fig. 2D, only myc-SEPT2 WT , but not myc-SEPT2 S218A , 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 Mg 2+ concentrations GTPcS replaced GDP efficiently, but could be readily stripped from the pro- tein during wash steps when the protein is bound on nickel beads in the low Mg 2+ concentrations due to its rapid GTPcS off-rate under these conditions. Using a filter-binding assay we measured both the equilibrium constant (K d ) and dissociation rate con- stant (k off ) of guanine nucleotides for SEPT2. To mon- itor GTP binding, a nonhydrolysable GTP analog, GTPcS, was used. Considering that Mg 2+ is known as Fig. 1. Septin 2 co-immunoprecipitates with several phospho- proteins. Immunoprecipitation with affinity purified anti-SEPT2 serum from cells labeled with [ 32 P]-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). GTPase properties of human SEPT2 Y W. Huang et al. 3250 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS an essential cofactor for many GTP-binding protein functions [27], we examined whether septin nucleotide binding and hydrolysis were affected by Mg 2+ concen- tration. Figure 5A,B represents the binding results of a set of independent experiments with different Mg 2+ concentrations for SEPT2. While binding of GTPcS was saturable at all Mg 2+ concentrations, it was clearly enhanced by Mg 2+ 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 Mg 2+ 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 K d for GTPcS was measured to be 0.28 ± 0.06 lm in 5mm MgCl 2 and 3.37 ± 1.42 lm in 0.01 mm MgCl 2 . Thus, the approximate 12-fold difference between low and high Mg 2+ concentrations indicates the import- ance of Mg 2+ in GTP-binding. Interestingly, this Mg 2+ dependence was not observed for GDP binding (Fig. 5B and Table 1). The K d values were very similar in the low micromolar range at different Mg 2+ con- centrations. Interestingly, when the nonphosphorylat- able S218A form of SEPT2 was examined, it had a much higher K d 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 GTP cS 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 Mg 2+ on the dis- sociation rate of GTPcS and GDP from SEPT2. AB 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). Y W. Huang et al. GTPase properties of human SEPT2 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3251 Figure 6A,B represents a set of independent experi- ments for the dissociation of GTPcS and GDP, respect- ively, from SEPT2 with different Mg 2+ 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-PackÒ C18 column. (B) A three molar ratio of GTPcS was added to the purified protein with 5 m M MgCl 2 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 mM 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 Mg 2+ . 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). GTPase properties of human SEPT2 Y W. Huang et al. 3252 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS From these panels we can see the same phenomenon as seen for the K d measurements, that at different Mg 2+ 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 Mg 2+ concentration. The summary of the k off 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 Mg 2+ is 1.24 min and in high Mg 2+ is 165 min. The dissociation half-life for GDP in low Mg 2+ is 3.62 min and that in high Mg 2+ is 23.1 min. Again, these results show Mg 2+ dependence for GTPcS disso- ciation, which has > 130-fold difference between low and high Mg 2+ concentrations while that for GDP has only a six-fold difference. In similar experiments carried out with the S218A mutant, we found that the k off for GTPcS and GDP at high Mg 2+ are not significantly different from those of SEPT2 WT (Table 2) with the dissociation half-life 182 min and 16.5 min for GTPcS and GDP, respectively, at high Mg 2+ concentration. Since the K d for GTPcS of the mutant is significantly increased, it implies that the association rate constant (k on ) decreases. In order to measure k on 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 k on and 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 (mM) GTPcS GDP Wild-type S218A Wild-type S218A K d (lM) nK d (lM) nK d (lM) nK d (lM) n 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 0.5 1.84 ± 0.1 0.93 ± 0.06 ND ND 1.19 ± 0.05 1.07 ± 0.1 ND ND 0.01 3.37 ± 1.42 0.70 ± 0.22 63.4 ± 10.8 0.73 ± 0.02 1.54 ± 0.37 1.02 ± 0.40 ND ND 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 Mg 2+ . Radioiso- topes used for GTPcS and GDP binding were 35 S-GTPcSand 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 experiments. ND, not done. MgCl2 (m M) GTPcS k off ( · 10 )3 s )1 ) GDP k off ( · 10 )3 s )1 ) WT S218A WT S218A 5 0.07 ± 0.02 0.06 ± 0.02 0.5 ± 0.1 0.7 ± 0.2 0.5 1.2 ± 0.1 ND 4.5 ± 1.6 ND 0.01 9.3 ± 2.5 ND 3.2 ± 0.8 ND Y W. Huang et al. GTPase properties of human SEPT2 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3253 SEPT2 steady-state kinetic constants, k cat and K m at different Mg 2+ concentrations. In Fig. 7, we show a representative experiment of SEPT2 at 5 mm Mg 2+ , 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 k cat and K m values are not significantly different at 5 and 0.5 mm MgCl 2 ; however, GTPase activity cannot be clearly measured without Mg 2+ in the buffer system. The k cat value for the SEPT2 S218A mutant is similar to that of the SEPT2 WT . However, consistent with the K d value, K m is 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 GTPc S 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 septin 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 mM MgCl 2 ,1mM EDTA, 5 mM 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 K m and k cat values represent mean ± SD of at least three independent experiments. The k cat s values were calculated from the V max values with the molecular mass of 43.5 kDa. NM, not meas- urable; ND, not done. MgCl 2 (mM) Wild-type S218A k cat (x10 )4 s )1 ) K m (lM) k cat ⁄ K m ( · 10 )4 s )1 ÆlM )1 ) k cat (x10 )4 s )1 ) K m (lM) k cat ⁄ K m ( · 10 )4 s )1 ÆlM )1 ) 5 2.7 ± 0.5 0.52 ± 0.05 5.2 4.6 ± 0.9 2.38 ± 0.08 1.9 0.5 2.5 ± 0.5 0.5 ± 0.04 5.0 ND ND ND 0NMNMNM NDNDND GTPase properties of human SEPT2 Y W. Huang et al. 3254 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS the 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 p21 H–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 K d of SEPT2 is 0.28 lm and that of GDP is 1.7 lm in the presence of physiological Mg 2+ concentrations. These values are about an order of magnitude lower than those observed for complexes of yeast septins expressed in bacteria. Complexes of Cdc3p-Cdc12p, Cdc3p-Cdc11p-Cdc12p and Cdc3p- Cdc10p-Cdc12p had K d values of 1.6, 6.2 and 5.9 for GTPcS. However, they are much more reminiscent of the K d 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 Mg 2+ -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. Y W. Huang et al. GTPase properties of human SEPT2 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3255 for GTPcS binding while GDP binding was Mg 2+ independent. Also, we made the surprising observation that the decay fitting models best fit either single- or bi-exponential curves depending on Mg 2+ 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 (t ½ ¼ 165 min) appears to be quite similar to that found for a SEPT2⁄ 6 ⁄ 7 complex expressed in bacteria (t ½ ¼ 150 min) [24]. This nucleotide binding property is not fully consistent with either Ras or Rho proteins. For example, p21 H–ras shows Mg 2+ -depend- ent guanine nucleotide binding behavior with a >500- fold difference in GDP k off in the presence and absence of Mg 2+ while Mg 2+ has no significant influence on K on [30,36,37]. In the case of Rho family proteins, RhoA, Cdc42 and Rac1 show similar K d values for GTPcS and GDP binding in the presence or absence of Mg 2+ although in the absence of Mg 2+ 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 Mg 2+ [34,35]. Unfortunately, due to our inability to produce SEPT2 in the nucleotide-free state, we were unable to measure k on and hydrolysis rate con- stants. The apparent k cat for SEPT2 as determined by steady state kinetics is very low (2.7 · 10 )4 s )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 K cat is similar to the intrinsic GTPase rate of p21 ras proteins (3.4–5 · 10 )4 s )1 ) [20,38] and that of Rho family members RhoA, RhoB, S. cerevisiae Cdc42 and Caenorhabditis elegans Cdc42 (3.4 · 10 )4 s )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 k cat also depends on Mg 2+ since it cannot be clearly measured following depletion of Mg 2+ , consis- tent with the fact that Mg 2+ is an essential cofactor for most GTPases. The crystal structure of RhoA revealed that elimination of the Mg 2+ 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 Mg 2+ -dependent and -independent binding properties of SEPT2 for GTPcS and GDP, respectively, may also indicate different conformations in recognizing triphos- phate and diphosphate guanine nucleotides. The simi- larity of the K d , k off and k cat of 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 pFast- GTPase properties of human SEPT2 Y W. Huang et al. 3256 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS BacHTb (Invitrogen, Burlington, ON, Canada) following PCR amplification. The SEPT2 S218A mutant was generated using the QuikChange TM 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 ⁄ P i ), pelleted again and kept at )80 °C for later use. The purification steps were carried out at 4 °C unless otherwise specified. Before purification, cell pellets from about 6 · 10 8 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 lgmL )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 1mm 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 of 1mgÆmL )1 at )80 °C and they were sta- ble for several months. In vivo [ 32 P]-orthophosphate labeling and immunoprecipitation SEPT2 WT or mutant SEPT2 S218A 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 SEPT2 WT or SEPT2 S218A using 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 [ 32 P]-orthophosphate for 5 h at 37 °C. 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, 1mm 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 l g) was added to 40 lL of 50% protein-A agarose beads (Invitrogen) and rotated for 1 h at 4 °C. 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 4 °C 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-SEPT2 WT 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 37 °C 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 4 °C for 30 min in buffer C (50 mm Tris ⁄ HCl, pH 8.0; 5mm b-mercaptoethanol; 0.04% Tween-20). The gels were preincubated in kinase buffer (40 mm Hepes, pH 7.5, Y W. Huang et al. GTPase properties of human SEPT2 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3257 [...]... 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GTP binding and hydrolysis kinetics of human septin 2 Yi-Wei Huang 1 , Mark C. Surka 1,3 , Denis Reynaud 2 , Cecil Pace-Asciak 2 and William