Abstract
Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)-beta isozymes. To better define this relationship, members of a library of recombinant Rho GTPases were screened for their capacity to directly engage various purified PLC-beta isozymes. Of the 17 tested members of the Rho family, only the active isoforms of Rac (Rac1, Rac2, and Rac3) both stimulate PLC-beta activity in vivo and bind PLC-beta2 and PLC-beta3, but not PLC-beta1, in vitro. Furthermore, the recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-beta2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases. Together, these findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC-beta isozymes and define a novel role for the PH domain of PLC-beta2 as a putative effector site for Rac GTPases.
Highlights
Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)- isozymes
The recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases
These findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC- isozymes and define a novel role for the PH domain of PLC-2 as a putative effector site for Rac GTPases
Summary
Protein Production—The coding sequences for all known human Rho family GTPases (Fig. 1A) were amplified by PCR and ligated into NcoI/XhoI-digested pProExHTa. PLC-␦ PH domain was purified similar to the above-described Rho GTPase purification. -Adrenergic receptor kinase 1 PH domain was produced and purified by a method similar to that used for the Rho GTPase purification. Insect cell-produced recombinant proteins were lysed using an Emulsi-Flex C5 (Avestin), extracted with 0.5% N-octyl--glucopyranoside, purified by immobilized metal ion affinity chromatography as described above for the Rho GTPases, and cleaved with TEV protease until the His tag was completely removed. Proteins were further purified using the above-described gel exclusion and ion exchange purification steps used for the PH domain of PLC-2. His6-tagged PLC-2 PH domain was attached to a CM5 chip (Biacore) coated with covalently attached anti-penta-histidine antibody (Qiagen) and washed with buffer to remove nonspecific binding before phospholipid injections were performed. The reaction was stopped after 1 h by aspiration of the medium and addition of 50 mM formic acid followed by neutralization with 150 mM NH4OH. [3H]Inositol phosphates were quantified by Dowex chromatography as described previously [36]
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