Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.
We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.
Krugmann
S
S
Inositide Laboratory, The Babraham Institute, Cambridge, CB2 4AT, United Kingdom.
Anderson
K E
KE
Ridley
S H
SH
Risso
N
N
McGregor
A
A
Coadwell
J
J
Davidson
K
K
Eguinoa
A
A
Ellson
C D
CD
Lipp
P
P
Manifava
M
M
Ktistakis
N
N
Painter
G
G
Thuring
J W
JW
Cooper
M A
MA
Lim
Z-Y
ZY
Holmes
A B
AB
Dove
S K
SK
Michell
R H
RH
Grewal
A
A
Nazarian
A
A
Erdjument-Bromage
H
H
Tempst
P
P
Stephens
L R
LR
Hawkins
P T
PT
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D011506:Proteins; D011994:Recombinant Proteins; D020741:rho GTP-Binding Proteins; C446512:ARAP3 protein, human; C060974:phosphatidylinositol 3,4,5-triphosphate; C000715348:ARF6 protein, human