In the present study, we show that the proline-rich domain of CdGAP is phosphorylated in vivo at multiple sites containing serine and threonine residues. We also demonstrate that CdGAP is phosphorylated downstream of the MEK-ERK pathway in response to serum or PDGF stimulation of fibroblasts. In particular, we find that CdGAP interacts with both ERK1/2 and RSK-1 and is directly phosphorylated by ERK-1 and RSK-1 in vitro. Site-directed mutagenesis reveals that threonine 776 of CdGAP is a phosphorylation site for ERK-1 and is an important regulatory site of CdGAP activity.
The incredibly large number of RhoGAP proteins strongly suggests a tight regulation of their activities at specific sites within the cell. Indeed, accumulating evidence indicates that RhoGAP activities are regulated by a wide variety of mechanisms, including phosphorylation. For example, tyrosine phosphorylation of p190RhoGAP by Src is necessary for its association with p120RasGAP and activation of its rhoGAP activity in vivo (11
). On the other hand, the in vitro GAP activity of RICS, a GTPase-activating protein for Cdc42 and Rac1, is inhibited by phosphorylation from Ca2+
/calmodulin-dependent protein kinase II (27
). Interestingly, MgcRacGAP, known to be involved in cytokinesis and a GAP for Rac1 and Cdc42, is functionally converted to a GAP for RhoA after serine phosphorylation by Aurora B kinase (20
). In the present study, we found that the replacement of threonine 776 by an alanine within the proline-rich domain of CdGAP is sufficient to induce a 1.5- and 2.0-fold increase in the in vitro and in vivo CdGAP activity toward Rac1, respectively. These findings suggest that phosphorylation of threonine 776 is an important regulatory site of CdGAP activity and may lead to a conformational change affecting the enzymatic activity. However, it is clear that CdGAP contains additional phosphorylation sites within the proline-rich domain that may affect the GAP activity as well. Future identification of these phosphorylation sites will help us to understand better the mechanism of regulation of CdGAP activity. In addition, it is more than likely that phosphorylation of the proline-rich domain may affect the localization of the protein or alter protein-protein interactions. Indeed, CdGAP contains five consensus SH3-binding sites. However, we have not yet been able to identify any SH3 domain-containing proteins binding to these motifs. Interestingly, Thr776
is located directly in the fifth proline-rich sequence PPLTPAPPPPT
P. Therefore, it is possible that phosphorylation of serine or threonine residues within the proline-rich domain causes a conformational change that negatively regulates their ability to bind SH3 domains. Similarly, it has been reported that phosphorylation of the proline-rich sequence of SOS is important to modulate its interaction with the SH3 domain-containing adaptor molecule Grb2 (7
To identify potential Ser/Thr kinases that interact with CdGAP, we performed an in-gel kinase assay. The success of this technique depends greatly on the ability of kinases to renature in the polyacrylamide gel. Several lines of evidence have demonstrated that RSK and ERK1/2 are able to efficiently recover their kinase activity after in-gel renaturation (8
). We found striking similarities between the in-gel kinase profile reported in these previous studies and our own results. In fact, we found that the two kinase activities recovered from the in-gel kinase are indeed ERK1/2 and RSK-1. Consistent with these results, we observed that CdGAP is present in a ternary protein complex including ERK1/2 and RSK-1. Moreover, we present evidence that the proline-rich domain of CdGAP is directly phosphorylated by ERK-1 and RSK-1 in vitro on distinct sites. These results suggest that both enzymes can phosphorylate CdGAP independently of each other. However, since RSK-1 phosphorylates CdGAP significantly less than ERK-1 in vitro, it remains to be determined whether prephosphorylation of CdGAP by ERK-1 or another kinase leads to a better substrate for RSK-1. It is also possible that efficient phosphorylation of CdGAP by RSK-1 requires the full-length protein and not only the proline-rich sequence. In fact, although the PRD domain of CdGAP contains a minimal RSK recognition phosphorylation site, the two additional ones in the central domain are preceded by arginine residues, which could make these sites more favorable for RSK-1. The levels of kinase activity obtained in the in-gel kinase experiment suggested that RSK-1 is more efficient than ERK-1 in phosphorylating the PRD of CdGAP, but the data obtained from the in vitro kinase assay with recombinant activated kinases suggests the opposite. These conflicting results can be explained by at least two possibilities. First, it is possible that RSK-1 recovers more efficiently its kinase activity after renaturation than ERK1/2. Second, RSK-1 seems to be more abundant than ERK-1/2 in coimmunoprecipitation assays with CdGAP.
In the present study, we have shown that the majority of the in vivo phosphorylation sites of CdGAP are on serine residues. Since RSK-1 interacts with and phosphorylates CdGAP on serine residues, it is likely that RSK-1 is responsible for most of the in vivo serine phosphorylation. In particular, treatment of Swiss 3T3 cells with the MEK inhibitor that blocks both ERK1/2 and RSK-1 activation also completely abolishes PDGF-induced CdGAP phosphorylation. We are currently investigating the role of RSK-1 phosphorylation on CdGAP, and future studies will provide valuable knowledge on this issue. Nevertheless, our studies clearly demonstrate that, although the extent of threonine phosphorylation in vivo is weak, the importance of this phosphorylation on CdGAP activity is significant.
The carboxy-terminal tail of CdGAP contains a number of putative ERK phosphorylation sites containing the consensus sequence P-X-S/T-P (10
). The in vitro and in vivo phosphopeptide mapping of CdGAP protein mutants strongly support the conclusion that ERK-1 phosphorylates CdGAP on Thr776
in vivo. However, amino acid substitution of both Thr769
to alanine did not completely abolish ERK-1 phosphorylation of CdGAP in vitro. In addition to the three ERK putative phosphorylation sites mutated in the present study, the proline-rich domain of CdGAP contains 12 S/T-P motifs containing the minimum consensus motif for ERK phosphorylation (33
). Interestingly, two of these sites are adjacent to a putative DEF domain containing the FPFP motif known to be an ERK docking site (12
). Indeed, mutation of this motif alters CdGAP binding to ERK1/2 and leads to a loss of CdGAP phosphorylation by ERK-1 in vitro.
CdGAP belongs to a novel family of RhoGAP proteins that are phylogenetically well conserved among different species. Up to three human genes encode for CdGAP-related proteins which consist of a RhoGAP domain at the N terminus and multiple SH3-binding motifs at the C terminus of the proteins. Interestingly, the DEF domain and the sequence surrounding Thr776 are found only in CdGAP and not in the closely related Grit or TCGAP. This suggests that ERK may exclusively interact with and phosphorylate CdGAP. In fact, among all of the characterized RhoGAP proteins, only mCdGAP and human DLC-1 proteins contain a DEF domain. Interestingly, we observed the differential expression of at least two major isoforms of CdGAP in specific mouse tissues. CdGAP-l (250 kDa) is predominantly expressed in the brain, lung, and heart, whereas CdGAP-s (90 kDa) is predominantly expressed in the liver and kidney. Whether the differential expression of CdGAP leads to a tissue-specific function for each isoform will require further investigation. We also found that both overexpressed CdGAP-s and CdGAP-l migrates higher than their expected molecular masses of 90 and 155 kDa, respectively. It will be of great interest to investigate the posttranslational modifications responsible for this impressive mobility shift.
Thus far, one of the most exciting roles attributed to RhoGAP proteins is their implication in the cross talk between members of the Rho family of small GTPases. For example, p120RasGAP interacts with and regulates p190RhoGAP activity, suggesting a possible interplay between Ras and Rho GTPases (25
). The connection between the Ras/MAPK pathway and the effects on cytoskeletal dynamics becomes more evident with the identification of a number of cytoskeleton-related proteins as ERK and RSK substrates (15
). Here we have demonstrated that phosphorylation of Thr776
by ERK affects CdGAP activity both in vitro and in vivo. One possibility is that mitogenic signal regulates Rac1 through phosphorylation and downregulation of CdGAP activity by ERK, leading to Rac1 activation and cytoskeletal remodeling (Fig. ). In conclusion, we provide evidence that CdGAP is a novel ERK substrate and may play roles in the connection between the Ras/MAPK and Rac1 pathways.
FIG. 10. CdGAP mediates cross talk between the Ras/MAPK pathway and the regulation of Rac1 activity. Upon PDGF stimulation, Ras activates the MAPK pathway, leading to gene expression and phosphorylation of many cytoplasmic and membrane proteins. In addition, Ras (more ...)