Rapid nucleotide exchange renders Asp-11 mutant actins resistant to depolymerizing activity of cofilin, leading to dominant toxicity in vivo.

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.

Cargo binding activates myosin VIIA motor function in cells.

Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility.

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA.

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca(2+) but not high Ca(2+). By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca(2+) suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.

Assessing transfer entropy from biochemical data.

We address the problem of evaluating the transfer entropy (TE) produced by biochemical reactions from experimentally measured data. Although these reactions are generally nonlinear and nonstationary processes making it challenging to achieve accurate modeling, Gaussian approximation can facilitate the TE assessment only by estimating covariance matrices using multiple data obtained from simultaneously measured time series representing the activation levels of biomolecules such as proteins. Nevertheless, the nonstationary nature of biochemical signals makes it difficult to theoretically assess the sampling distributions of TE, which are necessary for evaluating the statistical confidence and significance of the data-driven estimates. We resolve this difficulty by computationally assessing the sampling distributions using techniques from computational statistics. The computational methods are tested by using them in analyzing data generated from a theoretically tractable time-varying signal model, which leads to the development of a method to screen only statistically significant estimates. The usefulness of the developed method is examined by applying it to real biological data experimentally measured from the ERBB-RAS-MAPK system that superintends diverse cell fate decisions. A comparison between cells containing wild-type and mutant proteins exhibits a distinct difference in the time evolution of TE while any apparent difference is hardly found in average profiles of the raw signals. Such a comparison may help in unveiling important pathways of biochemical reactions.

Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors.

We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM-PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture.

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.

RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

p52Shc regulates the sustainability of ERK activation in a RAF-independent manner.

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.

Effect of phosphorylation in the motor domain of human myosin IIIA on its ATP hydrolysis cycle.

Previous findings suggested that the motor activity of human myosin IIIA (HM3A) is influenced by phosphorylation [Kambara, T., et al. (2006) J. Biol. Chem. 281, 37291-37301]; however, how phosphorylation controls the motor activity of HM3A is obscure. In this study, we clarify the kinetic basis of the effect of phosphorylation on the ATP hydrolysis cycle of the motor domain of HM3A (huM3AMD). The affinity of human myosin IIIA for filamentous actin in the presence of ATP is more than 100-fold decreased by phosphorylation, while the maximum rate of ATP turnover is virtually unchanged. The rate of release of ADP from acto-phosphorylated huM3AMD is 6-fold greater than the overall cycle rate, and thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form is markedly increased by phosphorylation by 30-fold. The dissociation constant for dissociation of the ATP-bound form of huM3AMD from actin is greatly increased by phosphorylation, and this result agrees well with the significant increase in the K(actin) value of the steady-state ATPase reaction. The rate constant of the P(i) off step is greater than 60 s(-1), suggesting that this step does not limit the overall ATP hydrolysis cycle rate. Our kinetic model indicates that phosphorylation induces the dissociation of huM3AMD from actin during the ATP hydrolysis cycle, and this is due to the phosphorylation-dependent marked decrease in the affinity of huM3AMD.ATP for actin and the increase in the ATP hydrolysis rate of huM3AMD in the actin-dissociated state. These results suggest that the phosphorylation of myosin IIIA significantly lowers the duty ratio, which may influence the cargo transporting ability of the native form of myosin IIIA that contains the ATP-independent actin binding site in the tail.

Crystallographic analysis reveals a unique conformation of the ADP-bound novel rice kinesin K16.

Biochemical studies revealed that the novel rice plant-specific kinesin K16 has several unique enzymatic characteristics as compared to conventional kinesins. The ADP-free form of K16 is very stable, whereas the ADP-free form of conventional kinesins is labile. In the present study, the crystal structure of the novel rice kinesin motor domain (K16MD) complexed with Mg-ADP was determined at 2.4 Å resolutions. The overall structure of K16MD is similar to that of conventional kinesin motor domains, as expected from the high amino acid sequence similarity (43.2%). However, several unique structures in K16 were observed. The position and length of the L5, L11, and L12 loops, which are key functional regions, were different from those observed in conventional kinesins. Moreover, the neck-linker region of the ADP-bound K16MD showed an ordered conformation at a position quite different from that previously observed in conventional kinesins. These structural differences may reflect the unique enzymatic characteristics of rice kinesin K16.

K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins.

Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion.

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells.

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral.

RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras-Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS-Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP-Ras/RalGDS clusters, to induce the full activation of Ral.

Intermolecular cross-linking of a novel rice Kinesin k16 motor domain with a photoreactive ATP derivative.

A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz(2)-epsilonATP was observed. Our results indicate that Bz(2)-epsilonATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.

Conformational change of the loop L5 in rice kinesin motor domain induced by nucleotide binding.

Loop L5 of kinesin is located near the ATPase site, in common with kinesins of various animal species. The rice plant-specific kinesin K16 also has a corresponding loop that is slightly shorter than that of mouse brain kinesin. The present study was designed to monitor conformational changes in loop L5 during ATP hydrolysis. For this purpose, we introduced one reactive cysteine into the L5 of rice kinesin and modified it with fluorescent probes. The cysteine in L5 was labeled with a fluorescent probe 2-(4'(iodoacetamide) anilino-naphthalene-6-sulfonic acid sodium salt) [IAANS]. IAANS was incorporated into L5 at an almost equimolar ratio in the absence of nucleotides. In contrast, the incorporated amount was reduced to 0.62 and 0.32 mol IAANS/mol motor domain in the presence of ATP and ADP, respectively. Upon nucleotide addition, the fluorescent intensity of IAANS incorporated into L5 was significantly reduced to 63% and 51% for ATP and ADP, respectively. These results suggest that L5 of rice kinesin significantly changes its conformation during ATP hydrolysis.

Preparation and characterization of a novel rice plant-specific kinesin.

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1.

Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein.

To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive.

Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 µM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.