Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.

Arcaro
A.
,
Wymann
M. P.
(
1993
).
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.
Biochem. J
296
,
297
301
Claesson-Welsh
L.
(
1994
).
Platelet-derived growth factor receptor signals.
J. Biol. Chem
269
,
32023
32026
Claesson-Welsh
L.
,
Eriksson
A.
,
Moren
A.
,
Severinsson
L.
,
Ek
B.
,
Östman
A.
,
Betsholtz
C.
,
Heldin
C.-H.
(
1988
).
cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.
Mol. Cell. Biol
8
,
3476
3486
Hansen
K.
,
Johnell
M.
,
Siegbahn
A.
,
Rorsman
C.
,
Engström
U.
,
Wernstedt
C.
,
Heldin
C.-H.
,
Rönnstrand
L.
(
1996
).
Mutation of a Src phosphorylation site in the PDGF-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.
EMBO J
15
,
5299
5313
Hawkins
P. T.
,
Eguinoa
A.
,
Qiu
R.-G.
,
Stokoe
D.
,
Cooke
F. T.
,
Walters
R.
,
Wennström
S.
,
Claesson-Welsh
L.
,
Evans
T.
,
Symons
M.
,
Stephens
L.
(
1995
).
PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.
Curr. Biol
5
,
393
403
Kumjian
D. A.
,
Wahl
M. I.
,
Rhee
S. G.
,
Daniel
T. O.
(
1989
).
Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C.
Proc. Nat. Acad. Sci. USA
86
,
8232
8236
Kundra
V.
,
Escobedo
J. A.
,
Kazlauskas
A.
,
Kim
H. K.
,
Rhee
S. G.
,
Williams
L. T.
,
Zetter
B. R.
(
1994
).
Regulation of chemotaxis by the platelet-derived growth factor receptor-.
Nature
367
,
474
476
Lim
L.
,
Manser
E.
,
Leung
T.
,
Hall
C.
(
1996
).
Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways.
Eur. J. Biochem
242
,
171
185
Nobes
C. D.
,
Hall
A.
(
1995
).
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia and filopodia.
Cell
81
,
53
62
Pawson
T.
(
1995
).
Protein modules and signalling networks.
Nature
373
,
573
580
Reif
K.
,
Nobes
C. D.
,
Thomas
G.
,
Hall
A.
,
Cantrell
D. A.
(
1996
).
Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways.
Curr. Biol
6
,
1445
1455
Rhee
S. G.
,
Suh
P.-G.
,
Ryu
S.-H.
,
Lee
S. Y.
(
1989
).
Studies of inositol phospholipid-specific phospholipase C.
Science
244
,
546
550
Ridley
A. J.
,
Hall
A.
(
1992
).
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70
,
389
399
Ridley
A. J.
,
Paterson
H. F.
,
Johnston
C. L.
,
Diekmann
D.
,
Hall
A.
(
1992
).
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70
,
401
410
Wahl
M.
,
Carpenter
G.
(
1991
).
Selective phospholipase C activation.
BioEssays
13
,
107
113
Wennström
S.
,
Hawkins
P.
,
Cooke
F.
,
Hara
K.
,
Yonezawa
K.
,
Kasuga
M.
,
Jackson
T.
,
Claesson-Welsh
L.
,
Stephens
L.
(
1994
).
Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling.
Curr. Biol
4
,
385
393
Wennström
S.
,
Siegbahn
A.
,
Yokote
K.
,
Arvidsson
A.-K.
,
Heldin
C.-H.
,
Mori
S.
,
Claesson-Welsh
L.
(
1994
).
Membrane ruffling and chemotaxis transduced by the PDGF-receptor require the binding site for phosphatidylinositol 3 kinase.
Oncogene
9
,
651
660
Yokote
K.
,
Margolis
B.
,
Heldin
C.-H.
,
Claesson-Welsh
L.
(
1996
).
Grb7 is a downstream signaling component of platelet-derived growth factor-and -receptors.
J. Biol. Chem
271
,
30942
30949
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