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rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain.

Walliser C, Retlich M, Harris R, Everett KL, Josephs MB, Vatter P, Esposito D, Driscoll PC, Katan M, Gierschik P, Bunney TD - J. Biol. Chem. (2008)

Bottom Line: 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.We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89070 Ulm, Germany.

ABSTRACT
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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Surface charge distribution of PLCγ1 and PLCγ2 spPHs. Surface electrostatic potentials representations of these two spPHs were computed with PyMol (top, PLCγ2 spPH; bottom, PLCγ1 spPH). Electrostatic potentials are represented as positive (blue), negative (red), and neutral (white) charges. The large loop that links the two parts of the spPHs (which is present in the published NMR structure of the PLCγ1 spPH) is not shown. The N view notation refers to the surface derived from the amino acid residues from the N-terminal half of the domain, and the C view refers to those residues derived from the C-terminal part.
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fig5: Surface charge distribution of PLCγ1 and PLCγ2 spPHs. Surface electrostatic potentials representations of these two spPHs were computed with PyMol (top, PLCγ2 spPH; bottom, PLCγ1 spPH). Electrostatic potentials are represented as positive (blue), negative (red), and neutral (white) charges. The large loop that links the two parts of the spPHs (which is present in the published NMR structure of the PLCγ1 spPH) is not shown. The N view notation refers to the surface derived from the amino acid residues from the N-terminal half of the domain, and the C view refers to those residues derived from the C-terminal part.

Mentions: The secondary structure elements of the spPHs from PLCγ1 and PLCγ2 align reasonably well (Fig. 4D) with a backbone root mean square difference of 2.9 Å over 65 core region Cα atoms. It is noteworthy that the sequence identity of the two spPHs is considerably lower (29%) than for the intact PLCγ1 and PLCγ2 proteins (49.5%) or the respective N-terminal PH domains (48%). Despite the relatively low sequence identity, when surface representations of the PLCγ2 and PLCγ1 spPH structures are compared (Fig. 5), there are clearly similarities. Overall, there is a strong correspondence of the surface distribution of charge and hydrophobicity. Notably, a patch of negative charge visible at the lower region of the N view is common between the two domains. Given this apparent global homology, it seems likely that the differences in Rac2 interaction with these two spPHs must reside in rather specific variation in surface side chain distribution. To probe this hypothesis, the residues important for the specific Rac2-binding interface were identified through NMR titration experiments and subsequent site-directed mutagenesis.


rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain.

Walliser C, Retlich M, Harris R, Everett KL, Josephs MB, Vatter P, Esposito D, Driscoll PC, Katan M, Gierschik P, Bunney TD - J. Biol. Chem. (2008)

Surface charge distribution of PLCγ1 and PLCγ2 spPHs. Surface electrostatic potentials representations of these two spPHs were computed with PyMol (top, PLCγ2 spPH; bottom, PLCγ1 spPH). Electrostatic potentials are represented as positive (blue), negative (red), and neutral (white) charges. The large loop that links the two parts of the spPHs (which is present in the published NMR structure of the PLCγ1 spPH) is not shown. The N view notation refers to the surface derived from the amino acid residues from the N-terminal half of the domain, and the C view refers to those residues derived from the C-terminal part.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2573054&req=5

fig5: Surface charge distribution of PLCγ1 and PLCγ2 spPHs. Surface electrostatic potentials representations of these two spPHs were computed with PyMol (top, PLCγ2 spPH; bottom, PLCγ1 spPH). Electrostatic potentials are represented as positive (blue), negative (red), and neutral (white) charges. The large loop that links the two parts of the spPHs (which is present in the published NMR structure of the PLCγ1 spPH) is not shown. The N view notation refers to the surface derived from the amino acid residues from the N-terminal half of the domain, and the C view refers to those residues derived from the C-terminal part.
Mentions: The secondary structure elements of the spPHs from PLCγ1 and PLCγ2 align reasonably well (Fig. 4D) with a backbone root mean square difference of 2.9 Å over 65 core region Cα atoms. It is noteworthy that the sequence identity of the two spPHs is considerably lower (29%) than for the intact PLCγ1 and PLCγ2 proteins (49.5%) or the respective N-terminal PH domains (48%). Despite the relatively low sequence identity, when surface representations of the PLCγ2 and PLCγ1 spPH structures are compared (Fig. 5), there are clearly similarities. Overall, there is a strong correspondence of the surface distribution of charge and hydrophobicity. Notably, a patch of negative charge visible at the lower region of the N view is common between the two domains. Given this apparent global homology, it seems likely that the differences in Rac2 interaction with these two spPHs must reside in rather specific variation in surface side chain distribution. To probe this hypothesis, the residues important for the specific Rac2-binding interface were identified through NMR titration experiments and subsequent site-directed mutagenesis.

Bottom Line: 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.We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89070 Ulm, Germany.

ABSTRACT
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.

Show MeSH