Kalirin is a GDP/GTP exchange factor (GEF) for Rho proteins that modulates the actin cytoskeleton in neurons. Alternative splicing generates Δ-isoforms, which encode the RhoGEF domain, but lack the N-terminal Sec14p domain and first 4 spectrin-like repeats of the full-length isoforms. Splicing has functional consequences, with Kal7 but not ΔKal7 causing formation of dendritic spines. Cells lacking endogenous Kalirin were used to explore differences between these splice variants. Expression of ΔKal7 in this system induces extensive lamellipodial sheets, while expression of Kal7 induces formation of adherent compact, round cells with abundant cortical actin. Based on in vitro and cell-based assays, Kal7 and ΔKal7 are equally active GEFs, suggesting that other domains are involved in controlling cell morphology. Catalytically inactive Kal7 and a Kalirin fragment which includes only Sec14p and spectrin-like domains retain the ability to produce compact, round cells and fractionate as high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PIP 2 (3,5) and PIP3, but does not alter cell morphology. We conclude that the Sec14p and Nterminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and Δ-Kalirin.

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.There are ϳ69 RhoGEFs in the human genome that are expected to catalyze nucleotide exchange on ϳ22 Rho GTPases (1). As the major activators of the Rho GTPases, guanine nucleotide exchange factor (GEF) 2 proteins play important roles in cell signaling, rearrangement of the cytoskeleton, membrane trafficking, and translational regulation (2). The widespread effects of GEF domains dictate the need for tight regulation of activity, which is underscored by the observation that deregulation of RhoGEFs is associated with cellular transformation and mental retardation (1, 3).Most RhoGEFs are composed of tandem Dbl homology (DH) and pleckstrin homology (PH) domains. RhoGEFs are regulated by cellular localization, interaction of phosphoinositides with the PH domain, tyrosine phosphorylation, oligomerization, and other protein/protein interactions (2, 4 -8). Although the interaction of phosphoinositides with the PH domain seems to be a general mechanism for modulating GEF activity (5, 6, 9 -12), other regulatory mechanisms affect a subset of RhoGEFs. RhoGEFs often contain several domains that are involved in their localization, association with other proteins, and regulation of GEF activity.Approximately one-third (ϳ22) of the human RhoGEFs contain Src homology 3 (SH3) domains (see Fig. 1). The GEFs with SH3 domains can be grouped into three classes based on the number and arrangement of the domains: Group I, with SH3 domains located N-terminally to the DH and PH domains; Group II, with SH3 domains located C-terminally; and Group III, with multiple SH3 domains. Several pieces of data support the hypothesis that the SH3 domains regulate GEF activity. For example, the cellular transforming activity of Ost is inhibited by its SH3 domain (13), and the first SH3 domain of Trio is necessary for GEFmediated effects on neurite outgrowth (14).Regulation of RhoGEFs by SH3 domains could be through an inhibitory intramolecular SH3 domain/ligand association. Most SH3 domains bind to a conse...

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction

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