Regulation of RhoGEF Activity by Intramolecular and Intermolecular SH3 Domain Interactions

Department of Molecular, Microbial and Structural Biology, University of Connecticut, Сторс, Connecticut, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2006; 281(27):18774-86. DOI: 10.1074/jbc.M512482200
Source: PubMed


RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22
human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the
enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions.
The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3
domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative
binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor
protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular
and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used
by other RhoGEF family members.

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    • "pGEX.GST-KGEF1, -KGEF1→7end and its mutants were purified as described (Alam et al., 1997; Penzes et al., 2001; Schiller et al., 2006). The pGEX-GST-Rac1 vector was a gift from Dr. Richard Cerione (Cornell University). "
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    ABSTRACT: Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.
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    ABSTRACT: The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.
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