Qiyu Feng

The cool-2/α-Pix protein mediates a Cdc42-Rac signaling cascade

Abstract Background: C loned- o ut o f l ibrary-2 (Cool-2)/ P AK- i nteractive e x change factor (α-Pix) was identified through its ability to bind the Cdc42/Rac target p 21- a ctivated k inase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. Results: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the D bl h omology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. Conclusions: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.

Cool-1 functions as an essential regulatory node for EGFreceptor- and Src-mediated cell growth

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool–1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v–Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.

Regulation of the Cool/Pix Proteins KEY BINDING PARTNERS OF THE Cdc42/Rac TARGETS, THE p21-ACTIVATED KINASES

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.

Novel regulatory mechanisms for the Dbl family guanine nucleotide exchange factor Cool-2/α-Pix

The Cool‐2 (cloned‐out of library‐2) protein (identical to α‐Pix for Pak‐interactive exchange factor) has been implicated in various biological responses including chemoattractant signaling and in certain forms of mental retardation. We show that when Cool‐2 exists as a dimer, it functions as a Rac‐specific guanine nucleotide exchange factor (GEF). Dimerization of Cool‐2 enables its Dbl (diffuse B‐cell lymphoma) and pleckstrin homology domains to work together (in trans) to bind specifically to Rac‐GDP. Dissociation of dimeric Cool‐2 into its monomeric form allows it to act as a GEF for Cdc42 as well as for Rac. The binding of either PAK (p21‐activated kinase) or Cbl (Casitas B‐lymphoma) to the SH3 domain of monomeric Cool‐2 is necessary for the functional interactions between GDP‐bound Cdc42 or Rac and the Cool‐2 monomer. The βγ subunit complex of large GTP‐binding proteins, by interacting with PAK, stimulates the dissociation of the Cool‐2 dimer and activates its GEF activity for Cdc42. Overall, these findings highlight novel mechanisms by which extracellular signals can direct the specific activation of Rac versus Cdc42 by Cool‐2/α‐Pix.

Tissue Transglutaminase Is an Essential Participant in the Epidermal Growth Factor-stimulated Signaling Pathway Leading to Cancer Cell Migration and Invasion

Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells.

Identification of a DOCK180-related Guanine Nucleotide Exchange Factor That Is Capable of Mediating a Positive Feedback Activation of Cdc42

Cdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an ∼220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity.

The Cbl proteins are binding partners for the Cool/Pix family of p21-activated kinase-binding proteins

Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21‐activated kinase (PAK). Using the yeast two‐hybrid screen, we identified Cbl‐b as a Cool family binding partner. We co‐immunoprecipitated endogenous Cool and Cbl‐b from a variety of breast cancer cell lines. The Cool–Cbl‐b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl‐b effectively blocks the ability of Cool‐2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl‐b acts as a negative regulator for signaling activities requiring PAK activation.

Phosphorylation of the Cool-1/β-Pix Protein Serves as a Regulatory Signal for the Migration and Invasive Activity of Src-transformed Cells

Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells.

Activation of the Ran GTPase Is Subject to Growth Factor Regulation and Can Give Rise to Cellular Transformation

Although the small GTPase Ran is best known for its roles in nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, recent studies have demonstrated the overexpression of Ran in multiple tumor types and that its expression is correlated with a poor patient prognosis, providing evidence for the importance of this GTPase in cell growth regulation. Here we show that Ran is subject to growth factor regulation by demonstrating that it is activated in a serum-dependent manner in human breast cancer cells and, in particular, in response to heregulin, a growth factor that activates the Neu/ErbB2 tyrosine kinase. The heregulin-dependent activation of Ran requires mTOR (mammalian target of rapamycin) and stimulates the capped RNA binding capability of the cap-binding complex in the nucleus, thus influencing gene expression at the level of mRNA processing. We further demonstrate that the excessive activation of Ran has important consequences for cell growth by showing that a novel, activated Ran mutant is sufficient to transform NIH-3T3 cells in an mTOR- and epidermal growth factor receptor-dependent manner and that Ran-transformed cells form tumors in mice.

A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies.