Matthew J. Hart

Downregulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK3β

Abstract Background: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor protein is responsible for both inherited and sporadic forms of colon cancer. Growth control by APC may relate to its ability to downregulate β -catenin post-translationally. In cancer, mutations in APC ablate its ability to regulate β -catenin, and mutations in β -catenin prevent its downregulation by wild-type APC. Moreover, signaling by the protein product of the wnt -1 proto-oncogene upregulates β -catenin and promotes tumorigenesis in mice. In a Xenopus developmental system, Wnt-1 signaling was inhibited by Axin, the product of the murine fused gene. This suggests a possible link between Axin, the Wnt-1 signaling components β -catenin and glycogen synthase kinase 3 β (GSK3 β ), and APC. Results: Human Axin (hAxin) binds directly to β -catenin, GSK3 β , and APC in vitro , and the endogenous proteins are found in a complex in cells. Binding sites for Axin were mapped to a region of APC that is typically deleted due to cancer-associated mutations in the APC gene. Overexpression of hAxin strongly promoted the downregulation of wild-type β -catenin in colon cancer cells, whereas mutant oncogenic β -catenin was unaffected. The downregulation was increased by deletion of the APC-binding domain from Axin, suggesting that APC may function to derepress Axin activity. In addition, hAxin dramatically facilitated the phosphorylation of APC and β -catenin by GSK3 β in vitro . Conclusions: Axin acts as a scaffold upon which APC, β -catenin and GSK3 β assemble to coordinate the regulation of β -catenin signaling.

The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell

Abstract Defects in β-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of β-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3β (GSK3β). Given that phosphorylation can regulate targeted degradation of β-catenin by the proteasome, β-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the β-catenin homolog Armadillo. We reasoned that the human homologs of Slimb – β-TrCP and its isoform β-TrCP2 (KIAA0696) – might interact with β-catenin. We found that the binding of β-TrCP to β-catenin was direct and dependent upon the WD40 repeat sequences in β-TrCP and on phosphorylation of the GSK3β sites in β-catenin. Endogenous β-catenin and β-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type β-TrCP in mammalian cells promoted the downregulation of β-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated β-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, β-TrCP2 did not associate with β-catenin. We conclude that β-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated β-catenin.

Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of alzheimer's disease

Background Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms [1], [2], [3], [4]. It was recently demonstrated that long-term treatment with rapamycin, an inhibitor of the mTOR pathway[5], or ablation of the mTOR target p70S6K[6] extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway would delay or prevent age-associated disease such as AD remained to be determined. Methodology/Principal Findings We used rapamycin administration and behavioral tools in a mouse model of AD as well as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Aβ42, a major toxic species in AD[7], in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR pathway can reduce Aβ42 levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy was increased in neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice, suggesting that the reduction in Aβ and the improvement in cognitive function are due in part to increased autophagy, possibly as a response to high levels of Aβ. Conclusions/Significance Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical settings, may be a potentially effective therapeutic agent for the treatment of AD.

IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

Proteins that associate with the GTP‐bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs‐GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP‐related domain (GRD) contained in IRA, NF‐1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C‐terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD‐dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co‐immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co‐immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co‐localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

A tyrosine-phosphorylated protein that binds to an important regulatory region on the cool family of p21-activated kinase-binding proteins

The p21-activated kinases (Pak) are major targets of the small GTPases Cdc42 and Rac. We, and others, recently identified a family of proteins termed Cool/Pix, which interact with Pak3. In cells, p50Cool-1 suppresses Pak activation by upstream activators; p85Cool-1 has a permissive effect on Pak activation, and we now show that the closely related Cool-2 stimulates Pak kinase activity. To understand the differential regulation of Pak by Cool proteins, we screened for Cool-interacting proteins by affinity purification and microsequencing. This has led to the identification of two closely related proteins called Cat (Cool-associated, tyrosine phosphorylated), which contain a zinc finger followed by three ankyrin repeats. Cat-1 is identical to the recently identified binding partner for the β-adrenergic receptor kinase (βARK or GRK-2), which was shown to have Arf-GAP activity. Cat-1 and Cat-2 both bind to the COOH-terminal region of p85Cool-1 and p85Cool-2but do not bind to p50Cool-1. Cat-1 is tyrosine-phosphorylated in growing NIH 3T3 fibroblasts, and its tyrosine phosphorylation is increased following cell spreading on fibronectin, decreased in cells arrested in mitosis, and increased in the ensuing G1 phase. Cat proteins are tyrosine-phosphorylated when co-expressed in cells with the focal adhesion kinase Fak and Src. These findings suggest that in addition to playing a role in Cool/Pak interactions, Cat proteins may serve as points of convergence between G protein-coupled receptors, integrins, Arf GTPases, cell cycle regulators, and Cdc42/Rac/Pak signaling pathways.

Identification of a Novel Guanine Nucleotide Exchange Factor for the Rho GTPase

The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.

Chronic inhibition of mammalian target of rapamycin by rapamycin modulates cognitive and non-cognitive components of behavior throughout lifespan in mice

Aging is, by far, the greatest risk factor for most neurodegenerative diseases. In non-diseased conditions, normal aging can also be associated with declines in cognitive function that significantly affect quality of life in the elderly. It was recently shown that inhibition of Mammalian TOR (mTOR) activity in mice by chronic rapamycin treatment extends lifespan, possibly by delaying aging {Harrison, 2009 #4}{Miller, 2011 #168}. To explore the effect of chronic rapamycin treatment on normal brain aging we determined cognitive and non-cognitive components of behavior throughout lifespan in male and female C57BL/6 mice that were fed control- or rapamycin-supplemented chow. Our studies show that rapamycin enhances cognitive function in young adult mice and blocks age-associated cognitive decline in older animals. In addition, mice fed with rapamycin-supplemented chow showed decreased anxiety and depressive-like behavior at all ages tested. Levels of three major monoamines (norepinephrine, dopamine and 5-hydroxytryptamine) and their metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid) were significantly augmented in midbrain of rapamycin-treated mice compared to controls. Our results suggest that chronic, partial inhibition of mTOR by oral rapamycin enhances learning and memory in young adults, maintains memory in old C57BL/6J mice, and has concomitant anxiolytic and antidepressant-like effects, possibly by stimulating major monoamine pathways in brain.

Identification of an Actin Cytoskeletal Complex That Includes IQGAP and the Cdc42 GTPase

The Rho subfamily of low molecular weight GTPases have been implicated in a variety of cellular functions that include reorganization of the actin cytoskeleton and stress-induced activation of the c-Jun kinase. The downstream targets that mediate the effects of Cdc42 on the actin cytoskeleton have yet to be fully identified. We have used the transient transfection of COS-7 cells with epitope-tagged Cdc42 to identify candidate signaling partners for this GTPase and identified the IQGAP protein as a major in vivo target for activated Cdc42. Epidermal growth factor stimulation of serum-starved COS-7 cells promoted the formation of a Cdc42-IQGAP complex, indicating that growth factors can increase the pool of activated Cdc42. Activated HA-Cdc42 co-localized with IQGAP or F-actin in vivo, whereas cells transfected with dominant-negative forms of Cdc42 (Cdc42T17N) showed predominantly dispersed distributions for both HA-Cdc42 and endogenous IQGAP. In detergent lysates from COS-7 cells transiently transfected with different forms of Cdc42, or from stably transfected CHO cells, the induction of actin polymerization by phalloidin resulted in the incorporation of both IQGAP and Cdc42 into actin-containing complexes. Taken together, these findings are consistent with a model whereby IQGAP serves as a target for GTP-bound Cdc42 providing a direct link between the activated GTPase and the actin cytoskeleton.

Guanine nucleotide exchange catalyzed by dbl oncogene product

Publisher Summary This chapter describes methods for the expression of Dbl and the Dbl homology (DH) domain in baculovirus expression systems and discusses the in vitro assays to demonstrate that Dbl and its DH domain contain guanine nucleotide exchange factors (GEFs) activity for the Cdc42Hs and Rho GTP-binding proteins. GEFs activate Ras family members of GTP-binding proteins by accelerating their rate of GDP release and, therefore, facilitating their GTP binding in cells. Unlike the case for Ras–GEFs for which the in vivo activities can be examined by the extent of conversion of Ras-bound GDP to GTP by the immunoprecipitation of Ras and thin-layer chromatography separation of the bound nucleotides, the GEF activities for Rho-type GTPases are difficult to determine in this manner because of the fast hydrolysis rates of the members of this family and the presence of abundant Rho–GDP-dissociation inhibitor (GDI), which renders most of the Rho-type proteins cytosolic and blocks the GEF action. Thus, the in vitro reconstituted assay is indispensable for detecting and quantitating the activities of potential Rho family GEF molecules.

Defects in the regulation of β-catenin in colorectal cancer

The molecular events that contribute to the progression of colon cancer are beginning to unravel. An initiating and probably obligatory event is the oncogenic activation of β-catenin. This can come about by the loss of its negative regulator the adenomatous polyposis coli (APC) protein, or by mutations in the β-catenin gene that result in a more stable protein product. The interaction between APC and β-catenin, and additional proteins that affect assembly and signaling along this pathway, are discussed.