Regulation of the Cool/Pix Proteins

The Cool (cloned-outof library)/Pix (forPAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. Three members of the Cool/Pix family have shown distinct regulatory activities: (i) p50 Cool-1 inhibits Cdc42/Rac-stimulated PAK activity, (ii) p85 Cool-1 /β-Pix has a permissive effect on Cdc42/Rac-stimulated activity, and (iii) p90 Cool-2 /α-Pix strongly activates PAK. We initially suspected that these different functional effects were due to a binding interaction that occurs at the carboxyl-terminal ends of the larger Cool/Pix proteins, thus enabling them to stimulate (or at least permit) rather than inhibit PAK activity. This led to the identification of the Cat proteins (forCool-associated tyrosine phosphosubstrates). However, here we show that the Cat proteins bind to the carboxyl-terminal ends of p85 Cool-1 (residues 523–546) and Cool-2 (residues 647–670), and that the binding of Cat to Cool-2 in fact is not necessary for the Cool-2-mediated activation of PAK. Rather, an 18-amino acid region, designated T1, that is present in the Cool-1 proteins, but missing in Cool-2, is essential for controlling the regulation of PAK activity by Cool-1/β-Pix in vivo. Deletion of T1 yielded a p85 Cool-1 molecule that mimicked the Cool-2 protein and was capable of strongly stimulating PAK activity. However, when T1 was added to Cool-2, the ability of Cool-2 to directly activate PAK was lost. We conclude that T1 represents a novel regulatory domain that accounts for the specific functional effects on PAK activity exhibited by the different members of the Cool/Pix family.

The small GTP-binding proteins Cdc42 and Rac serve as molecular switches in diverse biological response pathways, including the stimulation of cell cycle progression and gene transcription, alterations in the actin cytoskeleton, and the regulation of cell adhesion (1)(2)(3)(4)(5)(6)(7). A number of candidate downstream targets for Cdc42 and Rac have been identified, including the p21-activated serine/threonine kinases (PAKs, 1 Refs. 7-10), the activated Cdc42-associated tyrosine kinases (ACKs, Refs. 11 and 12), the p70 S6 kinase (13), IQGAPs (14 -16), and WASP (17)(18)(19). As the major targets of Cdc42 and Rac, the PAKs have been shown to trigger changes in gene expression as well as to mediate actin cytoskeletal and cell morphological changes (20 -24). In Saccharomyces cerevisiae, Ste20, the homolog of the mammalian PAKs, is involved in pheromone response by stimulating a mitogen-activated protein kinase cascade that leads to the transcription of genes required for cell cycle arrest as well as activates a distinct mitogen-activated protein kinase pathway that causes filamentous growth in response to nitrogen starvation (20). In mammalian cells, activated PAK mutants stimulate c-Jun kinase activity (7,21). Overexpression of PAK has also been shown to cause actin cytoskeletal rearrangements in a kinase-independent manner (25). Moreover, PAK is required for both Ras-mediated transformation (26) and Fas-mediated apoptosis (27,28). Overall, these findings demonstrate the importance of PAK in a number of different signaling pathways and emphasize the necessity of carefully regulating PAK activity.
A family of PAK-binding proteins that have strong regulatory effects on PAK function have been identified and called Cool (cloned-out of library) or Pix (PAK-interactive exchange factor) (29,30). We have been particularly interested in three members of the family, namely p50 Cool-1 , p85 Cool-1 (identical to ␤-Pix and the 85PR protein), and p90 Cool-2 (identical to ␣-Pix) (29 -32). In the present work, we set out to understand some initially puzzling results regarding the distinct functional effects exhibited by different members of the Cool/Pix family of proteins. When p50 Cool-1 was originally identified, it was suspected to serve as an upstream activator of Cdc42 or Rac because of the presence of a tandem DH/PH domain (29). The fact that the p50 Cool-1 protein was discovered based on its ability to directly bind PAK-3, a member of a family of Cdc42/ Rac-targets, thereby suggested the existence of a signaling complex that contained both an upstream regulator and a downstream effector for the Cdc42 or Rac GTP-binding protein. However, we found that p50 Cool-1 was not capable of stimulating PAK activity, as would have been expected if it were acting as a guanine nucleotide exchange factor for Cdc42 or Rac, but instead inhibited PAK activation by Dbl or by activated forms of Cdc42 (29). The p85 Cool-1 (␤-Pix) protein, which represents a longer splice variant form of p50 Cool-1 , did not inhibit Dbl-or activated Cdc42-stimulated PAK activity, but it also was incapable of directly stimulating PAK activity. However, the Cool-2 (␣-Pix) protein, which appears to represent a distinct gene product with SH3, DH, and PH domains that are ϳ70% identical to those found in the Cool-1 proteins, is able to strongly activate PAK. As an initial attempt to explain the distinct functional effects mediated by the different Cool/Pix proteins, we searched for potential cellular regulatory proteins that bound selectively to the extended carboxyl-terminal ends of p85 Cool-1 and Cool-2 and might prevent these carboxyl-terminal regions from adopting a conformation that inhibited access to the DH/PH domains and/or to the kinase active site of PAK. We in fact identified a family of specific binding partners for p85 Cool-1 and Cool-2, named Cats (Cool-associated tyrosine phosphosubstrates) (31), which are highly similar if not identical to proteins isolated through their abilities to bind to G protein-coupled receptor kinases (called Gits) and paxillin (called PKLs) (33,34). However, we show here that the Cats do not influence the ability of the Cool proteins to directly regulate PAK activity, although they may play a key role in the recruitment of Cool proteins to endosomal membranes. Instead, we have found that an 18-amino acid region, designated T1, serves as a novel and essential element for the regulation of PAK activation, accounting for the lack of stimulatory effects on PAK activity exhibited by p50 Cool-1 and p85 Cool-1 .
Protein Expression and Binding Assays-The different GST⅐p85Cool-1 constructs were each expressed in Escherichia coli and purified by glutathione-agarose affinity chromatography. Cell lysates from COS7 cells transiently transfected with Myc-or HA-tagged Cat-2 were combined with 20 l of a 1:1 suspension of GST fusion proteins bound to glutathione-Sepharose beads and incubated at 4°C for 2-3 h. The beads were then precipitated by microcentrifugation, washed three times with lysis buffer, and resuspended in 2ϫ SDS-PAGE sample buffer. Proteins were eluted by boiling for 5 min and separated on a 10% SDS-polyacrylamide gel, transferred to an Immobilon-P membrane and probed with mouse monoclonal anti-Myc or anti-HA antibodies. Primary antibodies were detected with horseradish peroxidase-coupled sheep anti-mouse antibody by ECL (Amersham Biosciences).
Immunoprecipitation and Western Blot Analysis-COS7 cells were washed with cold phosphate-buffered saline and lysed in buffer A (40 mM Tris-Cl, pH 7.4, 1% Triton, 100 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 10 g/ml leupeptin, and 10 g/ml aprotinin) plus 20 mM ␤-glycerophosphate and 5% glycerol. Lysates were centrifuged at 12,000 ϫ g for 15 min at 4°C. The cleared lysates were incubated with anti-PAK-3 primary antibody (a gift from Dr. S. Pelech, Kinetek Biotechnology Corporation, Vancouver, British Columbia) for 1 h followed by mixing with protein A-Sepharose beads for 45 min. The beads were washed three times with lysis buffer A and resuspended in Laemmli SDS-PAGE sample buffer. The immunoprecipitated proteins were resolved by 12% SDS-PAGE and transferred onto an Immobilon P membrane (Millipore). The membrane was immunoblotted with an anti-Myc antibody.
PAK Assays-Lysates were incubated with anti-PAK-3 primary antibody for 1.5 h followed by mixing with protein A-Sepharose beads (Invitrogen) for 45 min. The beads were washed three times with buffer A, twice with 2ϫ phosphorylation buffer (10 mM MgCl 2 and 40 mM Tris-HCl, pH 7.4), and mixed with 1.5 g/sample of the substrate myelin basic protein (Sigma). PAK assays were initiated by adding 25 M ATP and 10 Ci of [␥-32 P]ATP (3000 Ci/mmol) to the immunoprecipitation mixture and were performed at 22°C for 15 min. The reactions were stopped by adding 2ϫ SDS-PAGE sample buffer containing 20 mM EDTA. The phosphoproteins were resolved by SDS-PAGE (12% gel) and visualized by PhosphorImager analysis (Amersham Biosciences) prior to immunoblotting. The

RESULTS
Is Cat Binding Necessary for PAK Activity?-We have been studying three members of the Cool/Pix family in some detail; p50 Cool-1 , p85 Cool-1 , and Cool-2 ( Fig. 1). Given our working hypothesis that the binding of the Cat proteins to Cool-2 and/or p85 Cool-1 was responsible for their distinct functional effects relative to the inhibitory activity of p50 Cool-1 , we set out to delineate a limit-binding domain on the Cool proteins for the Cats and to test whether this binding interaction was in fact essential for the ability of Cool proteins to promote (or permit) rather than inhibit PAK activation. We began by considering three distinguishable regions within the carboxyl-terminal domain of p85 Cool-1 as potential Cat-binding sites; a histidine-rich domain (designated as 85HR, residues 419 -456), a proline-rich domain (PRD, residues 461-486), and a glutamic acid-rich domain (ERD, residues 511-634) ( Fig. 2A). To define which domain is capable of binding Cat, we prepared GST fusion proteins that contained each of these domains; specifically, GST⅐T1-HRD-PRD consisted of residues 394 -487 of p85 Cool-1 , GST⅐PR contained residues 460 -550, and GST⅐ER consisted of residues 511-640 ( Fig. 2A). The immobilized GST⅐T1-HRD-PRD, -PR, or -ER constructs were then incubated with cell lysates expressing hemagglutinin (HA)-tagged Cat-2. We found that both GST⅐PR and GST⅐ER were able to bind Cat (Fig. 2B).
There is an overlapping region shared by the GST⅐PR and Each of the Cool proteins contains SH3, DH, and PH domains. These domains are identical in p50 Cool-1 and p85 Cool-1 and are ϳ70% identical to the corresponding sequences found in Cool-2. Cool-2 additionally contains a calponin-homology (CH) domain, a serine-rich region (90SR), and proline-rich (PRD) and glutamic acid-rich regions (ERD) that share sequence similarity with regions in p85 Cool-1 . The p85 Cool-1 protein contains a distinct histidine-rich region (85HR) whereas p50 Cool-1 lacks this region but has a unique carboxyl-terminal domain (50C). Both p85 Cool-1 and p50 Cool-1 , but not Cool-2, contain an 18 amino acid region (designated T1) that connects the PH domain to 85HR or 50C.
GST⅐ER fusion proteins, representing residues 511-550 in p85 Cool-1 ( Fig. 2A). To better define the Cat-binding site, we made an additional four truncations within the ER domain, designated ER2, ER3, ER4 and ER5 ( Fig. 2A). As shown in Fig.  2B, the GST⅐ER2 and GST⅐ER3 fusion constructs had the same binding affinity for Cat as GST⅐ER, whereas the GST⅐ER4 construct was only capable of weak binding and GST⅐ER5 showed no detectable binding activity. These data suggest that the Cat-binding site lies within residues 527-542 on p85 Cool-1 .
To further delineate the Cat-binding site, we prepared another truncation, ER4.5 ( Fig. 2A), and found that like ER5, it was incapable of binding Cat (Fig. 2B). Taken together, these findings indicate that Cat binds between residues 527-539 on p85 Cool-1 (Fig. 2C). The amino acid sequence within this region is AALEEDAQILKVI. It should be noted that a highly con-served sequence is found within residues 685-698 of Cool-2 ( Fig. 2C).
To identify amino acid residues on p85 Cool-1 that are essential for binding Cat, we made four mutations within the GST⅐ER3 background (the individual point mutations L536A, K537A, V538A, and the triple mutation L536A/K537A/V538A. We expressed and purified these different GST⅐ER3 mutants and performed Cat-binding assays. We found that the [L536A]ER3 and [K537A]ER3 mutants were still fully capable of binding Cat (Fig. 3, lanes 2 and 3). However, both the [V538A]ER3 mutant and the [L536A/K537A/V538A]ER3 triple mutant showed little or no ability to bind Cat (Fig. 3, lanes 4  and 5). The same results were obtained when assaying p85 Cool-1 interactions with Cat in cells via the co-immunoprecipitation of these proteins (data not shown). Thus, Val-538 appears to be an essential residue for the binding of p85 Cool-1 to Cat.
We then examined whether the binding of Cat was in fact essential for the stimulatory effects mediated by Cool on PAK activity. We performed these experiments with Cool-2 because it is capable of strongly activating PAK, whereas p85 Cool-1 typically shows little detectable stimulation (29). Because we have found that the Cool-2 triple mutation L694A/K695A/ V696A does not completely eliminate its binding to Cat, to avoid any ambiguity a Cat-binding defective Cool-2 protein was generated by deleting the carboxyl-terminal end of the protein that includes the Cat-binding site (designated Cool-2(⌬641)). Wild type Cool-2 and the Cool-2(⌬641) mutant were co-transfected with HA-tagged Cat-2 into COS7 cells and binding assays were performed. The upper panel in Fig. 4A compares the relative levels of expression of the Cool-2 constructs. Under conditions where wild type Cool-2 gave a strong binding signal (Fig. 4A, lower panel, lane 2), the Cool-2(⌬641) deletion mutant was unable to bind Cat (lane 3).
We next directly compared the effects of these Cool-2 constructs on PAK activity. The upper panels in Fig. 4B compare the relative levels of expression of the Cool proteins being examined whereas the middle panels compare the relative amounts of PAK for the different assays. As earlier reported (31), we found that the expression of wild type Cool-2 was capable of strongly stimulating the ability of PAK to phosphorylate myelin basic protein, whereas p85 Cool-1 showed little stimulatory activity (Fig. 4B, lower left  and 3). The Cool-2(⌬641) truncation mutant, however, activated PAK to the same extent as the wild type Cool-2 protein (Fig. 4B, lower right panel, compare lanes 1 and 3), under experimental conditions where no binding of Cat was observed. These data clearly indicate that the binding of Cat is not required for the ability of Cool-2 to stimulate PAK activity.
If the binding of the Cat proteins is not a determinant for the stimulatory capability of Cool-2, then what role do Cool-Cat interactions play in Cool function? It has been reported that PAK-Cool-Cat complexes may be involved in vesicle trafficking because Cat contains an Arf-GAP activity and localizes to endosome membranes (35). Recently, we have found that although wild type Cool-2 co-localizes with endosomal markers (e.g. EEA1), Cool-2 mutants that are unable to bind Cat appeared to be more diffusely localized throughout the cytosol (data not shown). This leads us to suspect that Cool/Cat binding interactions are necessary for the proper translocation of Cool proteins to vesicle membranes.
Identification of a New Regulatory Domain on the Cool/Pix Proteins-Given the results described in the preceding section, we searched for other explanations for the distinct functional effects of the Cool/Pix proteins. We decided to consider possible differences between the Cool-1 and Cool-2 proteins, given that only Cool-2 is capable of directly activating PAK. Immediately downstream from the PH domain, Cool-2 contains a serine-rich region (designated as 90SR) whereas p85 Cool-1 contains a histidine-rich region (85 HR) (see Fig. 1). Both proteins then contain similar proline-rich (PRD) and carboxyl-terminal regions (ERD). Note that p50 Cool-1 lacks the histidine-rich region, as well as the proline-and glutamic acid-rich regions found in p85 Cool-1 , but contains a distinct carboxyl-terminal region (designated 50C). To examine the potential regulatory influence exerted by the carboxyl-terminal regions of p85 Cool-1 versus Cool-2, we prepared a carboxyl-terminal truncation mutant of Cool-1, designated SH⅐PH-Cool-1, which contains just the SH3, DH, and PH domains (Fig. 5A). This mutant was transiently transfected into COS7 cells and its effects on PAK activity were examined. In Fig. 5B, as well as others (i.e. Figs. 5C and 7), the upper panel compares the relative levels of expression of the different constructs being examined, whereas the middle panel compares the relative amounts of PAK-3 assayed. As expected, the Cool-2 protein gave rise to a strong stimulation of PAK activity, as assayed by the phosphorylation of an exogenous substrate (myelin basic protein) (Fig. 5B, lower panel, compare  lanes 2 and 5). Wild type p85 Cool-1 had no effect on PAK activity (compare lanes 1 and 5), consistent with previous findings (29); however, the carboxyl-terminal truncation mutant SH⅐PH-Cool-1 activated PAK as effectively as Cool-2 (lane 4). This suggested that there was an element downstream of the PH domain of p85 Cool-1 that negatively regulated PAK activation.
To more precisely define this negative regulatory region, we made another truncation mutant, designated SH⅐HR-Cool-1 (Fig. 5A), which lacks residues downstream of the 85HR domain. This truncation mutant behaved in a manner identical to wild type p85 Cool-1 and was unable to stimulate PAK activity (Fig. 5B, lower panel, compare lanes 2 and 3). Thus, these data indicated that the region in p85 Cool-1 that negatively regulates PAK activation is located between the PH and proline-rich domains.
Within this region of p85 Cool-1 , there are two distinguishable stretches of sequence. One is the histidine-rich region (85HR) that is absent from both p50 Cool-1 and Cool-2 (Fig. 5A). The other is immediately downstream from the PH domain and contains 18 amino acid residues that are conserved in p50 Cool-1 but not in Cool-2 (Fig. 5A). We have designated this region as T1. To define which of these two regions may be responsible for regulating PAK activity, we further truncated 85HR, yielding the construct designated SH⅐T1-Cool-1, and determined the effect of this truncation mutant on PAK activity. As shown in Fig. 5C (lower panel, compare lanes 3 and 4), SH⅐T1-Cool-1 activated PAK activity as effectively as Cool-2 (lower panel, lane 2), whereas SH⅐PR-Cool-1, which contains both 85HR and the proline-rich region (Fig. 5A), was completely ineffective. It should be noted that the inability of SH⅐HR-Cool-1 as well as SH⅐PR-Cool-1 to activate PAK was not because of their inability to bind PAK. Fig. 6 shows the results of experiments in which HA-tagged PAK-3 was co-expressed with different Myctagged Cool constructs, followed by precipitating PAK and Western blotting with anti-Myc antibody to detect associated Cool proteins. We found that both SH⅐HR-Cool-1 and SH⅐PR- Cool-1 bound PAK as effectively as Cool-2 and SH⅐T1-Cool-1, which strongly stimulate PAK activity. Thus, taken together, these results suggested that the 85HR region has an inhibitory effect on PAK activation.
Given that the p50 Cool-1 protein, which lacks the 85HR region, also is incapable of stimulating PAK activity but instead inhibits Dbl-or Cdc42-stimulated activity (29), we hypothesized that the T1 region may serve as a hinge that allows downstream regions on p85 Cool-1 (85HR) and p50 Cool-1 (e.g. the region designated 50C (Fig. 1)) to fold over and block access to the DH/PH domain, or the kinase domain of PAK, respectively. To test this idea, we took two approaches. First, we replaced the corresponding region of Cool-2 with the T1 region from Cool-1 and constructed the Cool-2 mutant designated Cool-2-T1-IN (Fig. 7A). If in fact T1 is responsible for mediating an inhibitory effect on PAK activation, then Cool⅐2-T1-IN should be incapable of stimulating PAK activity. Second, we replaced the entire carboxyl-terminal half of p85 Cool-1 , beginning with the T1 region and extending to the carboxyl terminus, with the corresponding region from Cool-2; this chimera was designated CHIM-Cool (Fig. 7A). Here the idea was that the absence of T1 should enable CHIM-Cool to activate PAK. In addition, we prepared two other Cool-2 truncation mutants, designated SH⅐PH-Cool-2 and SH⅐PR-Cool-2, that corresponded to the two Cool-1 truncation mutants SH⅐PH-Cool-1 and SH⅐PR-Cool-1, respectively (see Fig. 5A). Fig. 7B shows the results obtained with these different Cool constructs. The top panel compares the relative levels of expression of the different Cool proteins whereas the middle panel shows the relative amounts of PAK-3 for the different assays of PAK activity (bottom panel). As we expected, Cool-2-T1-IN lost the ability to stimulate PAK and behaved in a manner identical to p85 Cool-1 (Fig. 7B, lower panel, lane 6). On the other hand, CHIM-Cool, like Cool-2 (lanes 4 and 9), was able to strongly stimulate PAK activity (Fig. 7B, lower panel,  compare lanes 3 and 5). The truncation mutant, SH⅐PH-Cool-2, like SH⅐PH-Cool-1, stimulated PAK activity (Fig. 7B, lower  panel, see lanes 2 and 7). However, unlike SH⅐PR-Cool-1, which panel shows the relative amounts of PAK-3 assayed for each condition, and the lower panel shows the assay results, as described in the legend to Fig. 4. was not able to stimulate PAK activity, the SH⅐PR-Cool-2 truncation mutant lacking the T1 region was able to strongly activate PAK (Fig. 7B, lower panel, compare lanes 1 and 3). All of these data argue that the T1 region in the Cool-1 proteins is responsible for mediating the repression of PAK activation.
We went on to further examine whether the T1 region can mediate the inhibition of the guanine nucleotide exchange activity of p85 Cool-1 . As shown in Fig. 8, although the SH⅐PR-Cool-2 protein was capable of acting as a guanine nucleotide exchange factor (GEF), the chimera Cool-2-T1-IN (in which the T1 region from Cool-1 was inserted into SH⅐PR-Cool-2 immediately after the PH domain) did not catalyze nucleotide exchange. However, the SH⅐T1-Cool-1 truncation mutant did show GEF activity (Fig. 8), suggesting that the T1 region itself does not directly interact with the DH/PH domain and block its GEF activity. The GEF activities that we measured for the different Cool mutants correlated well with the effects of these mutants on PAK activation. Thus, taken together, these data support the idea that the T1 region is responsible for enabling downstream segments of the Cool proteins to inhibit GEF activity and PAK activation. DISCUSSION The Cool/Pix proteins contain the tandem arrangement of DH and PH domains that are characteristic of the Dbl family of guanine nucleotide exchange factors for Rho GTP-binding pro-teins. Because of this, their initial discovery as PAK-binding partners led to the assumption that the Cool/Pix proteins were upstream activators of Rac and/or Cdc42, directly complexed to a Cdc42/Rac target. However, it was soon appreciated that different members of the Cool/Pix family exhibited distinct functional effects on PAK such that p50 Cool-1 inhibited the ability of Dbl or activated Cdc42 to stimulate PAK activity, whereas p85 Cool-1 /␤-Pix did not interfere with Dbl-or Cdc42mediated stimulation, and Cool-2/␣-Pix strongly activated PAK (36). We originally questioned whether the distinct functional activities associated with the different Cool proteins were the outcome of a specific binding interaction between the carboxylterminal domains of p85 Cool-1 /␤-Pix and Cool-2/␣-Pix with a cellular protein that served as a regulatory factor. The idea was that because p50 Cool-1 lacked an extended carboxyl-terminal domain, it would not be capable of binding to the putative regulatory factor and thereby associated with PAK in an orientation that blocked access to the PAK active site. The binding of p85 Cool-1 /␤-Pix or Cool-2/␣-Pix to the regulatory factor would ensure the proper orientation of these Cool/Pix proteins such that they did not interfere with the kinase active site and instead permitted or even promoted PAK activation. This led to the identification of the Cat proteins, which are highly similar to the Git and PKL proteins (33,34), and bind to p85 Cool-1 /␤-Pix and Cool-2/␣-Pix but not to p50 Cool-1 . However, we show here quite clearly that the binding of Cat to Cool-2 is not necessary for Cool-2-mediated activation of PAK. Preliminary results from our laboratory suggest that the role of the Cats is to ensure the proper localization of the Cool/Pix proteins, particularly to endosomes. The ability of the Cool/Pix proteins to interact with the Cat/Git/PKL proteins, which in turn bind paxillin and function as Arf-GAPs, places them at focal adhesion complexes and endosomal membranes, perhaps providing a link between cell adhesion-induced signaling and intracellular trafficking. The necessity for such a link is suggested by the findings that Arf6, long suspected to be involved in intracellular trafficking, also controls cell spreading (37,38).
The finding that the Cats are not essential for Cool-2-medi- ated activation of PAK has led us to consider alternative mechanisms that could account for the functional differences between the different members of the Cool family. We have now delineated a limit region that accounts for the distinct functional activities. This 18 amino acid region (T1) contains 4 proline residues that may form a turn that enables downstream segments of p50 Cool-1 to fold back or become positioned in a way that blocks access to the active site of PAK or even prevents the guanine nucleotide exchange factor-independent activation of PAK that has been suggested to occur through the direct binding of the Cool-2/␣-Pix proteins (37). The T1 region also appears to account for the inability of p85 Cool-1 /␤-Pix to mirror the actions of Cool-2 and stimulate PAK activity, the idea being that T1 enables downstream segments of p85 Cool-1 to fold back over the DH/PH domains and inhibit guanine nucleotide exchange activity. The fact that the T1 region contains a number of serine and threonine residues (8 total) also raises the possibility that it serves as a site for regulatory phosphorylation events.
Various lines of evidence have now implicated the Cool/Pix proteins as playing key roles in interfacing diverse signaling pathways. The PAK proteins, which serve as primary binding partners for Cool/Pix, have also been implicated in a variety of biological activities ranging from integrin-mediated signaling and alterations in the actin cytoskeletal architecture to the activation of nuclear mitogen-activated protein kinases. Thus, PAK activation may serve to coordinate various cellular events and is also likely to require intricate regulation, as might be provided by the Cool/Pix proteins. Although we now have some insight into how the individual Cool proteins are able to confer distinct regulatory effects on PAK, a definitive understanding of the mechanisms underlying the negative regulatory activity of the T1 region will await structural determinations of the full-length Cool proteins. Moreover, little is known regarding to what extent each of the Cool/Pix proteins participate in the various functions of PAK. Future studies will be directed toward understanding whether multiple regulatory activities of Cool/Pix need to be exerted in the same cells or if different Cool proteins exert their specific regulatory effects in a cell-typespecific manner. It will also be interesting to see if the inhibitory effects exhibited by p50 Cool-1 on PAK activity, or the lack of stimulatory effects exhibited by p85 Cool-1 , can be altered or reversed by cellular proteins that bind to and/or phosphorylation events that alter the orientation of the T1 region.