ATPase and GTPase activities copurifying with GTP-binding proteins in E. coli

Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.     

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.     

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.     

GTP‐dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca²⁺‐mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 μM free Ca²⁺ causes maximum secretion—equivalent to that achieved with 100 μM free Ca²⁺—whereas GTPγS inhibits Ca²⁺‐stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP‐regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca²⁺. The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP‐dependent secretory apparatus requires at least one cytosolic protein. GTP‐dependent secretion can be reconstituted with crude HL‐60 and bovine liver cytosol. The reconstituting activity binds to GTP‐agarose, suggesting that the cytosolic factor is a GTP‐binding protein or forms a complex with a GTP‐binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion‐reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high‐molecular‐weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate‐ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP‐dependent secretion, and that activity from a BFA‐insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion. J. Cell. Biochem. 80:37–45, 2000. © 2000 Wiley‐Liss, Inc.     

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.     

Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.

beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.     

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt''s), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA^Phe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA^His and Phe-tRNA^Phe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt''s and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt''s.     

Domain structure and intramolecular regulation of dynamin GTPase.

Dynamin is a 100 kDa GTPase required for receptor-mediated endocytosis, functioning as the key regulator of the late stages of clathrin-coated vesicle budding. It is specifically targeted to clathrin-coated pits where it self-assembles into 'collars' required for detachment of coated vesicles from the plasma membrane. Self-assembly stimulates dynamin GTPase activity. Thus, dynamin-dynamin interactions are critical in regulating its cellular function. We show by crosslinking and analytical ultracentrifugation that dynamin is a tetramer. Using limited proteolysis, we have defined structural domains of dynamin and evaluated the domain interactions and requirements for self-assembly and GTP binding and hydrolysis. We show that dynamin's C-terminal proline- and arginine-rich domain (PRD) and dynamin's pleckstrin homology (PH) domain are, respectively, positive and negative regulators of self-assembly and GTP hydrolysis. Importantly, we have discovered that the alpha-helical domain interposed between the PH domain and the PRD interacts with the N-terminal GTPase domain to stimulate GTP hydrolysis. We term this region the GTPase effector domain (GED) of dynamin.     

Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins

The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.     

The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.     

Independence between GTPase active sites in the Escherichia coli cell division protein FtsZ

We have analyzed the substrate kinetics of the GTPase activity of FtsZ and the effects of two different GTPase inhibitors, GDP and the slowly hydrolyzable GTP analogue GMPCPP. In the absence of inhibitors the GTPase activity follows simple Michaelis-Menten kinetics, and both GDP and GMPCPP inhibited the activity in a competitive manner. These results indicate that the GTPase active sites in FtsZ filaments are independent of each other, a feature relevant to elucidate the role of GTP hydrolysis in FtsZ function and cell division.     

The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes

In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.     

Dimerization and its role in GMP formation by human guanylate binding proteins

The mechanism of oligomerization and its role in the regulation of activity in large GTPases are not clearly understood. Human guanylate binding proteins (hGBP-1 and 2) belonging to large GTPases have the unique feature of hydrolyzing GTP to a mixture of GDP and GMP with unequal ratios. Using a series of truncated and mutant proteins of hGBP-1, we identified a hydrophobic helix in the connecting region between the two domains that plays a critical role in dimerization and regulation of the GTPase activity. The fluorescence with 1-8-anilinonaphthalene sulfonate and circular dichroism measurements together suggest that in the absence of the substrate analog, the helix is masked inside the protein but becomes exposed through a substrate-induced conformational switch, and thus mediates dimerization. This is further supported by the intrinsic fluorescence experiment, where Leu²⁹⁸ of this helix is replaced by a tryptophan. Remarkably, the enzyme exhibits differential GTPase activities depending on dimerization; a monomer produces only GDP, but a dimer gives both GDP and GMP with stimulation of the activity. An absolute dependence of GMP formation with dimerization demonstrates a cross talk between the monomers during the second hydrolysis. Similar to hGBP-1, hGBP-2 showed dimerization-related GTPase activity for GMP formation, indicating that this family of proteins follows a broadly similar mechanism for GTP hydrolysis.     

Characterization of a GTPase-activating protein for the Ras-related Ral protein

We have demonstrated the presence of a GTPase-activating protein (GAP) for the Ras-related Ral A protein in the cytosolic fraction of brain and testis. This protein, designated Ral-GAP, was distinguished from Ras-GAP by its behavior in two chromatography systems and by the fact that the two GAP proteins did not stimulate the GTPase activity of each others target GTP binding proteins. The lack of effect of Ral-GAP on Ras GTPase activity also distinguished it from the product of the neurofibromatosis gene NF-1. Ral-GAP also differed from Rho-GAP and Rap-GAP by virtue of its elution from a gel filtration column with proteins of Mr greater than 10(6). This was likely an overestimate of the protein's molecular mass, however, since it sedimented in sucrose gradients between standard proteins of 150 and 443 kDa. Ral-GAP failed to promote the GTPase activity of mutant Ral proteins containing amino acid substitutions that in Ras lead to GAP-insensitive proteins.     

Insight into the GTPase activity of tubulin from complexes with stathmin-like domains

Microtubules are dynamically unstable tubulin polymers that interconvert stochastically between growing and shrinking states, a property central to their cellular functions. Following its incorporation in microtubules, tubulin hydrolyzes one GTP molecule. Microtubule dynamic instability depends on GTP hydrolysis so that this activity is crucial to the regulation of microtubule assembly. Tubulin also has a much lower GTPase activity in solution. We have used ternary complexes made of two tubulin molecules and one stathmin-like domain to investigate the mechanism of the tubulin GTPase activity in solution. We show that whereas stathmin-like domains and colchicine enhance this activity, it is inhibited by vinblastine and by the N-terminal part of stathmin-like domains. Taken together with the structures of the tubulin-colchicine-stathmin-like domain-vinblastine complex and of microtubules, our results lead to the conclusions that the tubulin-colchicine GTPase activity in solution is caused by tubulin-tubulin associations and that the residues involved in catalysis comprise the beta tubulin GTP binding site and alpha tubulin residues that participate in intermolecular interactions in protofilaments. This site resembles the one that has been proposed to give rise to GTP hydrolysis in microtubules. The widely different hydrolysis rates in these two sites result at least in part from the curved and straight tubulin assemblies in solution and in microtubules, respectively.     

Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson's disease R1441C/G mutants

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson's disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD.     

Phospholipids can switch the GTPase substrate preference of a GTPase-activating protein

The major cellular inhibitors of the small GTPases of the Ras superfamily are the GTPase-activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of GTPases, thereby inactivating them. The catalytic activity of several GAPs is reportedly inhibited or stimulated by various phospholipids and fatty acids in vitro, indicating a likely physiological role for lipids in regulating small GTPases. We find that the p190 RhoGAP, a potent GAP for the Rho and Rac GTPases, is similarly sensitive to phospholipids. Interestingly, however, several of the tested phospholipids were found to effectively inhibit the RhoGAP activity of p190 but stimulate its RacGAP activity. Thus, phospholipids have the ability to "switch" the GTPase substrate preference of a GAP, thereby providing a novel regulatory mechanism for the small GTPases.