Dataset Information

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester.

ABSTRACT: C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKC?, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKC? C1b (?C1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.

SUBMITTER: Geczy T

PROVIDER: S-EPMC3340006 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester.

Geczy Tamas T Peach Megan L ML El Kazzouli Saïd S Sigano Dina M DM Kang Ji-Hye JH Valle Christopher J CJ Selezneva Julia J Woo Wonhee W Kedei Noemi N Lewin Nancy E NE Garfield Susan H SH Lim Langston L Mannan Poonam P Marquez Victor E VE Blumberg Peter M PM

The Journal of biological chemistry 20120218 16

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologo ...[more]

PMID: 22351766

Similar Datasets

Project description:The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.

Dataset Information

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester.

Publications

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets