The pleckstrin homology domain of phospholipase D1 accelerates EGFR endocytosis by increasing the expression of the Rab5 effector, rabaptin-5.
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ABSTRACT:
SUBMITTER: Park MH
PROVIDER: S-EPMC4686693 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.
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Project description:Phospholipases C (PLCs) reversibly associate with membranes to hydrolyze phosphatidylinositol-4, 5-bisphosphate (PI[4,5]P(2)) and comprise four main classes: beta, gamma, delta, and epsilon. Most eukaryotic PLCs contain a single, N-terminal pleckstrin homology (PH) domain, which is thought to play an important role in membrane targeting. The structure of a single PLC PH domain, that from PLCdelta1, has been determined; this PH domain binds PI(4,5)P(2) with high affinity and stereospecificity and has served as a paradigm for PH domain functionality. However, experimental studies demonstrate that PH domains from different PLC classes exhibit diverse modes of membrane interaction, reflecting the dissimilarity in their amino acid sequences. To elucidate the structural basis for their differential membrane-binding specificities, we modeled the three-dimensional structures of all mammalian PLC PH domains by using bioinformatic tools and calculated their biophysical properties by using continuum electrostatic approaches. Our computational analysis accounts for a large body of experimental data, provides predictions for those PH domains with unknown functions, and indicates functional roles for regions other than the canonical lipid-binding site identified in the PLCdelta1-PH structure. In particular, our calculations predict that (1). members from each of the four PLC classes exhibit strikingly different electrostatic profiles than those ordinarily observed for PH domains in general, (2). nonspecific electrostatic interactions contribute to the membrane localization of PLCdelta-, PLCgamma-, and PLCbeta-PH domains, and (3). phosphorylation regulates the interaction of PLCbeta-PH with its effectors through electrostatic repulsion. Our molecular models for PH domains from all of the PLC classes clearly demonstrate how a common structural fold can serve as a scaffold for a wide range of surface features and biophysical properties that support distinctive functional roles.
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Project description:Mammalian phospholipase C-? (PLC-?) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how G?q and Rac1 bind PLC-?, there is a lack of consensus regarding the G?? binding site in PLC-?. Using FRET between cerulean fluorescent protein-labeled G?? and the Alexa Fluor 594-labeled PLC-? pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal G?? binding region in PLC-?3. We show that the isolated PH domain can compete with full-length PLC-?3 for binding G?? but not G?q, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-? PH domain as the G?? binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-?, necessitating conformational rearrangement to allow G?? binding. Blocking PH domain motion in PLC-? by cross-linking it to the EF hand domain inhibits stimulation by G?? without altering basal activity or G?q response. The fraction of PLC-? cross-linked is proportional to the fractional loss of G?? response. Cross-linked PLC-? does not bind G?? in a FRET-based G??-PLC-? binding assay. We propose that unliganded PLC-? exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-? modulates G?? access to its binding site.
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Project description:Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5'-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22-Rabex-5-Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.
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Project description:Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also unclear are observations that the extent of Gbetagamma activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of the PLCbeta2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to that with Gbetagamma subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic headgroups. Also, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gbetagamma activatable mutant, PLCbeta2/delta1(C193S). We find an increase in the level of FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral headgroups. These results suggest that enzymatic activation can be conferred through optimal association of the PHbeta71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gbetagamma activation on different membrane surfaces.
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Project description:Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP(2) in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.
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