Identification of ARAP3, a Novel PI3K Effector Regulating Both Arf and Rho GTPases, by Selective Capture on Phosphoinositide Affinity Matrices

Krugmann, Sonja; Anderson, Karen E.; Ridley, S.; Risso, N.; McGregor, Alex; Coadwell, John; Davidson, Keith; Eguinoa, Alicia; Ellson, Chris D.; Lipp, Peter; Manifava, Maria; Ktistakis, Nicholas T.; Painter, Gavin F.; Thuring, Jan Willem; Cooper, Matthew A.; Lim, Ze-Yi; Holmes, Andrew B.; Dove, Stephen K.; Michell, Robert H.; Grewal, Anita; Nazarian, Arpi; Erdjument‐Bromage, Hediye; Tempst, Paul; Stephens, Len R.; Hawkins, Phillip T.

doi:10.1016/s1097-2765(02)00434-3

Cited by 286 publications

(327 citation statements)

References 45 publications

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“…PI3K phosphorylates PI(4,5)P 2 to create PI(3,4,5,)P 3 [33] and SHIP2 is a negative regulator of the PI3K pathway [17] that dephosphorylates PI(3,4,5)P 3 lipids to PI(3,4)P 2 [15]. Importantly, as shown previously by us, Arap3 binds PI(3,4,5,)P 3 stronger than it binds PI(3,4)P 2 [3]. Since binding of Arap3 to PI(3,4,5)P 3 is required for efficient membrane localization of Arap3, dephosphorylation of PI(3,4,5,)P 3 by SHIP2 implies a reduced affinity of Arap3 for the plasma membrane.…”

Section: Discussionsupporting

confidence: 64%

“…Rabbit polyclonal anti-SHIP2 antibody and sheep anti-Arap3 antibody were described before [3,15]. Where indicated, cells were stimulated with 20 ng/ml EGF (ICN Biomedicals Inc.), 1 μg/ml insulin (Sigma) and 10 μM LY294002 (Sigma).…”

Section: Antibodies and Reagentsmentioning

confidence: 99%

“…GFP-ΔSAMArap3 (residues 71-1544) was made using mutagenesis PCR with GFP-Arap3 [3] as a template. FlagHis-tagged Arap3 was created using Gateway Technology (Invitrogen).…”

Section: Plasmids and Constructsmentioning

confidence: 99%

“…Arap3 (Arf GAP, Rho GAP, Ankyrin repeat and PH domains) is a PI3K effector protein that was first identified through its ability to bind PI(3,4,5,)P 3 lipids [3]. Upon binding of PI(3,4,5,)P 3 lipids to one of its pleckstrin homology (PH) domains, Arap3 translocates to the plasma membrane and is activated to serve as a dual GTPase activating protein (GAP) for Arf and Rho G-proteins [4].…”

Section: Introductionmentioning

confidence: 99%

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The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

Raaijmakers

Deneubourg

Rehmann

et al. 2007

Cellular Signalling

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Arap3 is a phosphoinositide (PI) 3 kinase effector that serves as a GTPase activating protein (GAP) for both Arf and Rho G-proteins. The protein has multiple pleckstrin homology (PH) domains that bind preferentially phosphatidyl-inositol-3,4,5-trisphosphate (PI(3,4,5,)P 3 ) to induce translocation of Arap3 to the plasma membrane upon PI3K activation. Arap3 also contains a Ras association (RA) domain that interacts with the small G-protein Rap1 and a sterile alpha motif (SAM) domain of unknown function. In a yeast two-hybrid screen for new interaction partners of Arap3, we identified the PI 5′-phosphatase SHIP2 as an interaction partner of Arap3. The interaction between Arap3 and SHIP2 was observed with endogenous proteins and shown to be mediated by the SAM domain of Arap3 and SHIP2. In vitro, these two domains show specificity for a heterodimeric interaction. Since it was shown previously that Arap3 has a higher affinity for PI(3,4,5,)P 3 than for PI(3,4)P 2 , we propose that the SAM domain of Arap3 can function to recruit a negative regulator of PI3K signaling into the effector complex.

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Section: Discussionsupporting

confidence: 64%

Section: Antibodies and Reagentsmentioning

confidence: 99%

“…GFP-ΔSAMArap3 (residues 71-1544) was made using mutagenesis PCR with GFP-Arap3 [3] as a template. FlagHis-tagged Arap3 was created using Gateway Technology (Invitrogen).…”

Section: Plasmids and Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

Raaijmakers

Deneubourg

Rehmann

et al. 2007

Cellular Signalling

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“…Cytosolic fractions were prepared from COS-7 cells transfected with Myc-LL5␤ and mixed with Affi-Gel beads covalently attached to PtdIns(3,4,5)P 3 (16). The beads were washed, and bound proteins were eluted and immunoblotted with anti-Myc antibody.…”

Section: Ll5␤ a Pip 3 And ␥-Filamin-binding Proteinmentioning

confidence: 99%

LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

Paranavitane¹,

Coadwell²,

Eguinoa³

et al. 2003

Journal of Biological Chemistry

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We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5␤). The protein bound PtdIns(3,4,5)P 3 selectively in vitro. Strikingly, a substantial proportion of LL5␤ became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P 3 levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5␤ to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5␤ that could not bind PtdIns(3,4,5)P 3 were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P 3 levels, LL5␤ was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns-(3,4,5)P 3 levels, it became significantly plasma-membrane localized. The distribution of LL5␤ is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P 3 indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P 3 . In addition, LL5␤ bound to the cytoskeletal adaptor, ␥-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed ␥-filamin with GFP-LL5␤ in the unidentified vesicles.Phosphoinositide 3-kinases (PI3Ks) 1 3-phosphorylate phosphoinositides. There are three classes of PI3Ks. The type I enzymes seem to act as PtdIns(4,5)P 2 3-kinases in vivo; they can be activated by a variety of close-to-receptor transduction events and drive accumulation of PtdIns(3,4,5)P 3 in the inner leaflet of the plasma membrane. This PtdIns(3,4,5)P 3 serves as signal recruiting proteins from the cytosol that possess modules, typically PH domains, capable of binding its head group (1).There are a variety of reagents that can be used to inhibit PI3K activity. Most widely used are wortmannin (2) and LY294002 (3); both of which potently inhibit nearly all classes of PI3K and hence cannot generally be used to implicate a particular PI3K in a process. More specific are receptor tyrosine kinase Tyr 3 Phe mutants. A number of receptor tyrosine kinases (relevant here, the PDGF ␤-receptor) are capable of binding type IA PI3Ks at specific tyrosine residues that become phosphorylated following ligand binding (4). Mutation of these tyrosines to phenylalanine blocks type IA PI3K binding and activation but does not affect association of other effectors (1, 5). Stable, clonal cell lines have been created overexpressing wild-type PDGF-␤ receptors or (Y740F/Y751F) PDGF-␤ receptors (6) that have allowed the impact of selectivity blocking type IA PI3K activation to be assessed in vivo (7).There is now a substantial famil...

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Pleckstrin Homology (PH) Domains

Shaw

2002

Encyclopedia of Molecular Biology

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Identification of ARAP3, a Novel PI3K Effector Regulating Both Arf and Rho GTPases, by Selective Capture on Phosphoinositide Affinity Matrices

Cited by 286 publications

References 45 publications

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

Pleckstrin Homology (PH) Domains

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