DOCK180 and its orthologs are essential activators of various Rac GTPase-dependent biological processes 14–17,21,23
. We and others have demonstrated that DOCK180 promotes GTP-loading of Rac through an atypical GEF domain termed the DHR-2 domain 21,23
. We report here that another conserved domain in DOCK180-related proteins, DHR-1, is also indispensable for the biological function of DOCK180, as it couples the production of PtdIns(3,4,5)P3
at the membrane to DOCK180-mediated Rac GTP loading.
We found that forms of DOCK180 which either lack DHR-1 or contain point mutations in that domain fail to induce cell elongation and directional cell movement. The DOCK180 DHR-1 protein was found to induce Rac GTP-loading to the same extent as the wild-type DOCK180, supporting the notion that GTP-loading of Rac alone is not sufficient to induce processes that require cell polarization 36
. Instead, it is known that formation of a PtdIns(3,4,5)P3
gradient at the membrane mediates Rac-dependent actin reorganization at the leading edge, which is required for directional cell movement 4–6
. What had remained unclear is how PtdIns(3,4,5)P3
integrates with DOCK180 signaling to regulate Rac function upon cell polarization. We report here that the DHR-1 domain directly binds to PtdIns(3,4,5)P3
. Furthermore, a form of DOCK180 in which the DHR-1 domain had been replaced by a canonical PtdIns(3,4,5)P3
binding PH domain efficiently mediated cell elongation and cell migration. Thus, the two conserved domains in DOCK180, DHR-1 and DHR-2, function to integrate PtdIns(3,4,5)P3
signaling with Rac GTP-loading, and this integration is essential for membrane polarization and cell migration to occur.
Our finding that DOCK180 translocates to the plasma membrane in response to PtdIns(3,4,5)P3
production is in agreement with a report by Kobayashi et al. 37
. These authors suggested that a cluster of basic amino acids in the carboxyl-terminus of DOCK180 could mediate an ionic interaction with phosphate residues of PtdIns(3,4,5)P3
. Kobayashi et al.
noted, however, that this ionic interaction is not sufficient for the binding of full-length DOCK180 to PtdIns(3,4,5)P3
(which inhibits an ionic interaction) cannot compete with PtdIns(3,4,5)P3
binding to full-length DOCK180 37
. Thus, region(s) other than the basic domain must participate in the recognition of PtdIns(3,4,5)P3
and DOCK180 membrane translocation. Our studies identify the DHR-1 domain as this region since we found that it binds to PtdIns(3,4,5)P3
in a non-ionic manner, and that it is essential for PtdIns 3-kinase-induced membrane translocation of DOCK180.
The structural basis of the DHR-1 domain-mediated PtdIns(3,4,5)P3
binding is presently unknown. Identification of a putative C2 domain within the DHR-1 domain is interesting, but requires experimental verification. C2 domains typically interact with lipids such as phosphatidylserine and phosphatidylcholine: we failed to detect such interactions for DHR-1. Recently, the C2 domains of JFC1 and Rsb5 proteins have been reported to bind rather promiscuously to several phosphoinositides, including PtdIns(3,4,5)P3 26,28
. Therefore, subsets of C2 domains are emerging as genuine phosphoinositide-binding modules. Our finding that point mutations in the putative CBR1 and CBR3 loops in the DHR-1 domain abolish both the lipid binding activity and the function of the protein provide supporting evidence for the presence of a C2-like domain within DHR-1. Since the sequences of the DHR-1 domain are conserved across the DOCK180-related proteins, it is plausible that the lipid binding function is also conserved. Indeed, in vitro
experiments demonstrate that several other DHR-1 domains [those of DOCK3, DOCK6 and DOCK9 (zizimin1)] also have lipid-binding activity (not shown).
Our results and previous studies by others demonstrate that the presence of all three components of the CrkII-ELMO-DOCK180 complex is required for cell elongation and migration 15,21,23
. Our studies here provide a molecular explanation for the role of DOCK180 in this complex, that of coupling PtdIns(3,4,5)P3
signaling with Rac GTP-loading. Going forward, it will be important to identify the biological events regulated by this multiprotein complex that are PtdIns(3,4,5)P3
-dependent. Also of note, our studies do not rule out the possibility that ELMO1 and CrkII could provide membrane targeting activity. For example, it has been suggested that the PH domain of Ced-12 (ELMO) is required for membrane targeting and protein function in C. elegans 17
. We have also detected lipid binding activity in the PH domain of ELMO (not shown), but further studies will be required to determine the specificity of this domain and the biological function of this activity. Additional work is needed to understand the full biological significance of the CrkII-ELMO-DOCK180 protein complex and its regulation in cellular signaling.