Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex.

The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.

A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with "off-target" molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions.

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

The Bucentaur (BCNT) protein family: a long-neglected class of essential proteins required for chromatin/chromosome organization and function.

The evolutionarily conserved Bucentaur (BCNT) protein superfamily was identified about two decades ago in bovines, but its biological role has long remained largely unknown. Sparse studies in the literature suggest that BCNT proteins perform important functions during development. Only recently, a functional analysis of the Drosophila BCNT ortholog, called YETI, has provided evidence that it is essential for proper fly development and plays roles in chromatin organization. Here, we introduce the BCNT proteins and comprehensively review data that contribute to clarify their function and mechanistic clues on how they may control development in multicellular organisms.

Rasa3 controls megakaryocyte Rap1 activation, integrin signaling and differentiation into proplatelet.

Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.

Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing ß1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.

Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.

Multiple duplication of the bucentaur gene family, which recruits the APE-like domain of retrotransposon: Identification of a novel homolog and distinct cellular expression.

The p97bcnt/cfdp2 is a ruminant-specific gene created by a combination of gene duplication of ancestral bcnt (bucentaur) or cfdp1 (craniofacial developmental protein 1), bcnt/cfdp1, and the insertion of a retrotransposable element-1 (RTE). As a result, p97Bcnt recruits the whole apurinic/apyrimidinic endonuclease (APE)-like domain of RTE in the middle of the molecule (RTE domain) as a region encoded by an exon. In addition, p97Bcnt contains two intramolecular repeats (IRs) of 40 amino acids each in the C-terminal region, whereas Bcnt/Cfdp1 contains one IR. We have identified an additional bovine homolog with a structure highly similar to p97Bcnt, designated p97Bcnt2, which contains three IRs. p97bcnt2 is located in tandem with bcnt/cfdp1 and p97bcnt within a 177-kb range on bovine chromosome 18, a syntenic region of human chromosome 16. The gene product is expressed as a protein with an apparent molecular mass of 102 kDa. The phylogenetic tree strongly suggests that p97bcnt-2 forms a third clade of the bcnt family and that the first duplication of the IR unit occurred prior to the divergence of p97bcnt and p97bcnt-2. To address the question of whether these bcnt members have distinct functions, we first examined the expression and localization of the p97Bcnt family members. p97Bcnt is substantially expressed in many tissues involved in responses to external and internal stress. In the testis, p97Bcnt localizes preferentially in the nuclei of spermatozoa, while Bcnt/Cfdp1 localizes predominantly in the cytosol of Leydig cells and some spermatogenic cells, implying that at least these two molecules of the Bcnt family play different functional roles. These results provide evidence for the direct contribution of RTE to gene diversity to form gene families that may support cellular function.

RasGAPs: a crucial regulator of extracellular stimuli for homeostasis of cellular functions.

Ras and its GTPase activating proteins (GAPs) are among the crucial regulators of extracelluar ligands. Information about these regulators has been elucidated during the course of studies in signal transduction over the last two decades. RasGAPs such as p120GAP and neurofibromin have been studied extensively for their roles as either "negative" regulators or effectors of Ras. Accumulating evidence suggests that these molecules are crucial regulators of extracellular stimuli that serve to maintain the homeostasis of cellular functions. This compendium highlights cellular functions of RasGAPs and their signaling characteristics from the viewpoint of homeostasis, including our recent finding of the phenotype of R-RasGAP mutant mice whose GAP activity is down-regulated.

Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1.

The members of the transcription factor Foxo family regulate the expression of genes concerned with the stress response, cell cycle and gluconeogenesis. Foxo1 (FKHR) contains 15 consensus phosphorylation sites for the mitogen-activated protein kinase (MAPK) family. Therefore, we hypothesized that MAPKs could directly regulate the transcriptional activity of Foxo1 via phosphorylation. In vitro kinase assay showed that Foxo1 was phosphorylated by extracellular signal-regulated kinase (Erk) and p38 MAPK (p38) but not by c-jun NH2-terminal kinase (JNK). In NIH3T3 cells, epidermal growth factor or anisomycin increased phosphorylation of exogenous Foxo1, which was significantly inhibited by pretreatment with an MEK 1 inhibitor, PD98059, or a p38 inhibitor, SB203580. Two-dimensional phosphopeptide mapping using mutation of phosphorylation sites for MAPK revealed that the nine serine residues in Foxo1 are specifically phosphorylated by Erk and that five of the nine residues are phosphorylated by p38 in vivo. Moreover, we also found that Foxo1 interacts with Ets-1 and functions as a coactivator for Ets-1 on the fetal liver kinase (Flk)-1 promoter in bovine carotid artery endothelial cells. Mutation of the nine phosphorylation sites for Erk in Foxo1 was shown to lead to less binding and synergistic activity for Ets-1 on the Flk-1 promoter when compared with wild-type Foxo1. These results suggest that Foxo1 is specifically phosphorylated by Erk and p38, and that this phosphorylation regulates the function of Foxo1 as a coactivator for Ets-1.

Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development.

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

A tandem gene duplication followed by recruitment of a retrotransposon created the paralogous bucentaur gene (bcntp97) in the ancestral ruminant.

Retrotransposable element-1 (RTE-1) is a class of long interspersed nucleotide elements that contain in its open reading frame an apurinic/apyrimidinic endonuclease domain (AP-END) and a reverse transcriptase domain. Ruminants have a clade-specific RTE-1 (BovB/RTE). The bovine bcnt gene (bucentaur or craniofacial developmental protein 1) has a duplicated paralog (bcntp97) in tandem that recruited an AP-END of BovB/RTE as a coding exon (RTE exon). We obtained sequence of the bcnt region from several animals and showed that other ruminants also have the bcntp97 with a conserved RTE exon while camels and pigs do not. Genomic Southern analysis showed that camels and pigs have multiple bcnt-related sequences but not BovB/RTE which bovines and lesser mouse deer have abundantly. These results indicate that the bcnt gene duplication followed by the creation of bcntp97 including recruitment of the RTE exon occurred in the ancestral ruminant about 55 MYA. The indication of time frame is supported by a phylogenetic analysis. Taken together with a result of differential tissue expression of the two bcnt paralogs, we conclude that bcntp97 was created concurrently with the early radiation of BovB/RTE in an ancestral ruminant and then acquired a novel function.

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons.

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons.

Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells.

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.

A novel Giraffidae-specific interspersed repeat with a microsatellite, originally found in an intron of a ruminant paralogous p97bcnt gene.

The ruminant-specific p97bcnt gene (bcntp97) is a paralogous gene that includes a region derived from a retrotransposable element 1 (RTE-1). The region comprises an exon (RTE-1 exon) encoding 325 amino acids in the middle of the p97bcnt protein. To understand how the bcntp97 paralog evolved, we examined its organization in several ruminants. We found a 700-base pair (bp) insert in the 5' intron of the RTE-1 exon in giraffe bcntp97. This insert is missing in the corresponding regions of bovine and sika deer. Furthermore, the sequence of the insert is interspersed in the genome of giraffe but not bovine and also contains a (GA)n microsatellite. A highly homologous insert harboring significantly different (GA)n microsatellite was detected in the corresponding region of okapi bcntp97. Therefore, the interspersed fragments with (GA)n microsatellite might serve as a marker for tracking how duplicated genes evolve in a family-specific manner.

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts.

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.

A transposable element-mediated gene divergence that directly produces a novel type bovine Bcnt protein including the endonuclease domain of RTE-1.

Ruminant Bcnt protein with a molecular mass of 97 kDa (designated p97Bcnt) includes a region derived from the endonuclease domain of a retrotransposable element RTE-1. Human and mouse Bcnt proteins lack the corresponding region but have a highly conserved 82-amino acid region at the C-terminus that is not present in p97Bcnt. By screening a bovine BAC library, we found two more bcnt-related genes: human-type bcnt (h-type bcnt) and its processed pseudogene. Whereas the pseudogene is localized on chromosome 26, both bcntp97 and the h-type bcnt genes are found on bovine chromosome 18, a synteny region of human chromosome 16 on which human BCNT is localized. Complete nucleotide sequencing of the BAC clone reveals that the bcntp97 and h-type bcnt genes are located just 6 kb apart in a tandem manner. The two h-type bcnt and bcntp97genes are active at both the transcriptional level and the protein level. H-type bovine Bcnt is more like human BCNT than p97Bcnt, when compared at their N-terminal regions. However, phylogenetic analysis using the N-terminal region of the bcnt gene family revealed that the duplication of bovine genes occurred within the bovine lineage with significantly accelerated substitution in bcntp97. This acceleration was not ascribed definitely to positive selection. After duplication, one of the bovine bcnt genes recruited the endonuclease domain of an intronic RTE-1 repeat accompanied by the accelerated substitution at the 5'-ORF, resulting in creation of a novel type of Bcnt protein in bovine.

The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains.

There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C-terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol-binding activity as the full-size toxin with structural stability. Cell membrane-bound recombinant domain 4 was detected in the floating low-density fractions and was found to be cofractionated with the raft-associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol-rich rafts. Furthermore, an enhanced green fluorescent protein-domain 4 fusion protein stains membrane surfaces in a cholesterol-dependent manner in living cells. Therefore, domain 4 of theta-toxin is an essential cholesterol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.