Despite the critical roles of Dbl family GEFs in mitogenic signaling, the mechanisms that regulate their activity are incompletely understood. Especially enigmatic are the mechanisms that maintain GEF activity “in check” prior to, and following, cell stimulation. Here, we describe the identification and characterization of a novel regulatory mechanism for the guanine nucleotide exchange factor Dbl. This pathway includes the molecular chaperones Hsc70 and Hsp90 and the U-box ubiquitin-protein ligase CHIP. We show that through interactions with these regulators, the proto-oncogenic form of Dbl is efficiently ubiquitinated and rapidly degraded, resulting in low steady-state expression levels of the protein. Importantly, we show that the oncogenic form of Dbl “escapes” regulation by the Hsc70/Hsp90/CHIP complex.
For many Dbl family members, mutations outside the catalytic DH/PH domain cause constitutive activity and elevated oncogenic potential. In the case of Dbl and its homologs Vav, Ect2, Tiam1, Asef, and Net1, oncogenic activation results from the deletion of sequences in the amino terminus of the proto-oncogene (10
). Thus, it appears that regions in the amino terminus have an inhibitory function that attenuates the GEF's catalytic activity. In support of this notion, it has been reported that coexpression of the isolated amino terminus of Dbl inhibits the GEF activity of oncogenic Dbl, suggesting an intramolecular, autoinhibitory mode of regulation (5
). However, the molecular basis of this regulation is likely to be more complex, since the autoinhibited state could not be reconstituted in vitro with purified components (5
). Our findings indicate that the amino terminus of proto-Dbl attenuates the protein's GEF activity by binding the regulators of ubiquitination and degradation.
It was previously reported that the oncogenic form of the Dbl protein degrades at a lower rate than its proto-oncogenic counterpart (24
). The basis for this difference, however, remained unknown. We show here that, through its amino-terminal spectrin domain, proto-Dbl binds the molecular chaperones Hsc70 and Hsp90 and the ubiquitin ligase CHIP and that these molecules regulate the levels, the localization, and the activity of proto-Dbl. These interactions render proto-Dbl a short-lived, aggregation-prone protein, the fate of which is dictated by the triage decision, stabilization versus degradation. While Hsp90 dictates stabilization of proto-Dbl, CHIP directs the protein to ubiquitination and subsequent degradation (Fig. ). Hence, these interactions maintain the steady-state expression of proto-Dbl at low levels and comprise an efficient mechanism for keeping the protein's activity in check. Oncogenic Dbl, lacking the spectrin domain association interface, cannot bind the mediators of the triage decision. The result of the oncogenic truncation mutation, therefore, is that onco-Dbl escapes degradation and accumulates in cells at high levels. This increase in expression levels leads to persistent activation of the substrate GTPases and their downstream pathways, ultimately causing cell transformation. In that respect, onco-Dbl resembles many other growth regulatory molecules that transform cells by virtue of their elevated protein levels.
A schematic model of the regulation of Dbl proteins by ubiquitination and proteasomal degradation. See Discussion for details.
The role that ubiquitin-mediated degradation plays in the response of proto-Dbl- expressing cells to external stimuli is presently unknown. In view of the present observations, it is tempting to speculate that the activation of proto-Dbl by cell surface receptors will influence its association with the (co)chaperones and thus modulate its rate of degradation (Fig. ). Verification of this point awaits further experimental examination.
The interactions that regulate the ubiquitination and degradation of proto-Dbl are different from those that control the fate of other growth-promoting molecules that are subject to the triage decision. For example, in the case of p53, Akt, B-Raf, the growth factor receptor ErbB2/Neu, and v-Src, inhibition of Hsp90 results in selective degradation of the mutated, oncogenic variant of the protein (3
). Consequently, Hsp90 inhibitors are presently used to treat malignancies in which the mutated, oncogenic protein is expressed (2
). In the case of Dbl, the situation is reversed: only the proto-oncogenic form of the protein is subject to downregulation, while the constitutively active form evades this fate. These observations suggest that malignancies in which truncated versions of Dbl-related GEFs are expressed will be resistant to treatment with inhibitors of Hsp90 function.
The molecular chaperones Hsc70 and Hsp90 have been shown to modulate the intracellular levels and activities of a number of signaling molecules, including the GEFs FGD1 (30
), CNRasGEF (51
), and Vps9p (15
). The present study describes a new Hsp90/Hsc70 client protein, the guanine nucleotide exchange factor proto-Dbl, suggesting the possibility that other proteins that bear structural and functional homology may be similarly regulated.