To understand how phosphatidylinositol 3-kinase (PI3K) modulates cell structure and function, we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik1(Delta)2(Delta)) missing two (DdPIK1 and 2) of three PI3K genes, which are homologues of the mammalian p110 subunit. Levels of [32P]phosphatidylinositol 3, 4 bisphosphate (PI(3,4)P2) and [32P]phosphatidylinositol trisphosphate (PIP3) were reduced in pik1(Delta)2(Delta), which had major defects in morphological and functional correlates of macropinocytosis. This was accompanied by dramatic deficits in a subset of F-actin-enriched structures such as circular ruffles, actin crowns and pseudopodia. Although pik1(Delta)2(Delta) were mobile, they failed to aggregate into streams. Therefore we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI(3,4)P2, regulate the organization of actin filaments necessary for circular ruffling during macropinocytosis, the extension of pseudopodia and the aggregation of cells into streams, but not the regulation of cell motility.

REFERENCES

Adams
R. J.
,
Pollard
T. D.
(
1989
).
Binding of myosin I to membrane lipids.
Nature
340
,
565
568
Araki
N.
,
Johnson
M. T.
,
Swanson
J. A.
(
1996
).
A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages.
J. Cell Biol
135
,
1249
1260
Bokoch
G. M.
,
Vlahos
C. J.
,
Wang
Y.
,
Knaus
U. G.
,
Traynor-Kaplan
A. E.
(
1996
).
Rac GTPase interacts specifically with phosphatidylinositol 3-kinase.
Biochem. J
315
,
775
759
Browning
D. D.
,
The
T.
,
O'Day
D. H.
(
1995
).
Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP.
Cell Signal
7
,
481
489
Buczynski
G.
,
Grove
B.
,
Nomura
A.
,
Kleve
M.
,
Bush
J.
,
Firtel
R. A.
,
Cardelli
J.
(
1997
).
Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport and actin cytoskeleton organization.
J. Cell Biol
136
,
1271
1286
Burgering
B. M.
,
Coffer
P. J.
(
1995
).
Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction [see comments].
Nature
376
,
599
602
Cohen
C. J.
,
Bacon
R.
,
Clarke
M.
,
Joiner
K.
,
Mellman
I.
(
1994
).
Dictyostelium discoideum mutants with conditional defects in phagocytosis.
J. Cell Biol
126
,
955
966
Ding
J.
,
Vlahos
C. J.
,
Liu
R.
,
Brown
R. F.
,
Badwey
J. A.
(
1995
).
Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils.
J. Biol. Chem
270
,
11684
91
Dynes
J. L.
,
Clark
A. M.
,
Shaulsky
G.
,
Kuspa
A.
,
Loomis
W. F.
,
Firtel
R. A.
(
1994
).
LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.
Genes Dev
8
,
948
58
Franke
J.
,
Kessin
R.
(
1977
).
A defined minimal medium for axenic strains of Dictyostelium discoideum.
Proc. Nat. Acad. Sci. USA
74
,
2157
2161
Franke
T. F.
,
Kaplan
D. R.
,
Cantley
L. C.
,
Toker
A.
(
1997
).
Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate [see comments].
Science
275
,
665
668
Gerisch
G.
,
Hess
B.
(
1974
).
Cyclic-AMP-Controlled oscillations in suspended Dictyostelium cells: their relation to morphogenetic cell interactions.
Proc. Nat. Acad. Sci. USA
71
,
2118
2122
Hadwiger
J. A.
,
Firtel
R. A.
(
1992
).
Analysis of G alpha 4, a G-protein subunit required for multicellular development in Dictyostelium.
Genes Dev
6
,
38
49
Hawkins
P. T.
,
Eguinoa
A.
,
Qiu
R. G.
,
Stokoe
D.
,
Cooke
F. T.
,
Walters
R.
,
Wennstrom
S.
,
Claesson-Welsh
L.
,
Evans
T.
,
Symons
M.
, et al. 
(
1995
).
PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.
Curr. Biol
5
,
393
403
Hu
P.
,
Margolis
B.
,
Skolnik
E. Y.
,
Lammers
R.
,
Ullrich
A.
,
Schlessinger
J.
(
1992
).
Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.
Mol. Cell. Biol
12
,
981
90
Hu
Q.
,
Klippel
A.
,
Muslin
A. J.
,
Fantl
W. J.
,
Williams
L. T.
(
1995
).
Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase.
Science
268
,
100
102
Jackson
S. P.
,
Schoenwaelder
S. M.
,
Matzaris
M.
,
Brown
S.
,
Mitchell
C. A.
(
1995
).
Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form ofphosphoinositide 3-kinase.
EMBO J
14
,
4490
500
Joly
M.
,
Kazlauskas
A.
,
Fay
F. S.
,
Corvera
S.
(
1994
).
Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites.
Science
263
,
684
687
Jung
G.
,
Wu
X.
,
Hammer
J. A.
3rd
(
1996
).
Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions [see comments].
J. Cell Biol
133
,
305
323
Kapeller
R.
,
Chakrabarti
R.
,
Cantley
L.
,
Fay
F.
,
Corvera
S.
(
1993
).
Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3kinase complexes: potential interactions with the microtubule cytoskeleton.
Mol. Cell. Biol
13
,
6052
6063
Kimura
K.
,
Hattori
S.
,
Kabuyama
Y.
,
Shizawa
Y.
,
Takayanagi
J.
,
Nakamura
S.
,
Toki
S.
,
Matsuda
Y.
,
Onodera
K.
,
Fukui
Y.
(
1994
).
Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase.
J. Biol. Chem
269
,
18961
18967
Klippel
A.
,
Kavanaugh
W. M.
,
Pot
D.
,
Williams
L. T.
(
1997
).
A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.
Mol. Cell. Biol
17
,
338
344
Kodaki
T.
,
Woscholski
R.
,
Emr
S.
,
Waterfield
M. D.
,
Nurse
P.
,
Parker
P. J.
(
1994
).
Mammalian phosphatidylinositol 3-kinase induces a lethal phenotype on expression in Schizosaccharomyces pombe; comparison with the VPS34 gene product.
Eur. J. Biochem
219
,
775
780
Kotani
K.
,
Hara
K.
,
Kotani
K.
,
Yonezawa
K.
,
Kasuga
M.
(
1995
).
Phosphoinositide 3-kinase as an upstream regulator of the small GTP-binding protein Rac in the insulin signaling of membrane ruffling.
Biochem. Biophys. Res. Commun
208
,
985
990
Kotani
K.
,
Yonezawa
K.
,
Hara
K.
,
Ueda
H.
,
Kitamura
Y.
,
Sakaue
H.
,
ando
A.
,
Chavanieu
A.
,
Calas
B.
,
Grigorescu
F.
, et al. 
(
1994
).
Involvement of phosphoinositide 3-kinase in insulin-or IGF-1-induced membrane ruffling.
EMBO J
13
,
2313
2321
Kozma
R.
,
Ahmed
S.
,
Best
A.
,
Lim
L.
(
1995
).
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
Mol. Cell. Biol
15
,
1942
1952
Lu
P. J.
,
Shieh
W. R.
,
Rhee
S. G.
,
Yin
H. L.
,
Chen
C. S.
(
1996
).
Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity.
Biochemistry
35
,
14027
14034
Mann
S. K.
,
Firtel
R. A.
(
1991
).
A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium.
Mech. Dev
35
,
89
101
Michiels
F.
,
Habets
G. G.
,
Stam
J. C.
,
van der Kammen
R. A.
,
Collard
J. G.
(
1995
).
A role for Rac in Tiam1-induced membrane ruffling and invasion.
Nature
375
,
338
340
Nakanishi
H.
,
Brewer
K. A.
,
Exton
J. H.
(
1993
).
Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem
268
,
13
16
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
Novak
K. D.
,
Peterson
M. D.
,
Reedy
M. C.
,
Titus
M. A.
(
1995
).
Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.
J. Cell Biol
131
,
1205
1221
O'Halloran
T. J.
,
Anderson
R. G.
(
1992
).
Characterization of the clathrin heavy chain from Dictyostelium discoideum.
DNA Cell Biol
11
,
321
330
O'Konski
M. S.
,
Pandol
S. J.
(
1990
).
Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell.
J. Clin. Invest
86
,
1649
1657
Ostap
E. M.
,
Pollard
T. D.
(
1996
).
Overlapping functions of myosin-I isoforms? [comment].
J. Cell Biol
133
,
221
224
Qiu
R. G.
,
Chen
J.
,
Kirn
D.
,
McCormick
F.
,
Symons
M.
(
1995
).
An essential role for Rac in Ras transformation.
Nature
374
,
457
459
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
Rivero
F.
,
Koppel
B.
,
Peracino
B.
,
Bozzaro
S.
,
Siegert
F.
,
Weijer
C. J.
,
Schleicher
M.
,
Albrecht
R.
,
Noegel
A. A.
(
1996
).
The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility and contribute to phagocytosis and development.
J. Cell Sci
109
,
2679
2691
Singh
S. S.
,
Chauhan
A.
,
Brockerhoff
H.
,
Chauhan
V. P.
(
1993
).
Activation of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.
Biochem. Biophys. Res. Commun
195
,
104
112
Singleton
C. K.
,
Delude
R. L.
,
Ken
R.
,
Manning
S. S.
,
McPherson
C. E.
(
1991
).
Structure, expression and regulation of members of the developmentally controlled V and H gene classes from Dictyostelium.
Dev. Genet
12
,
88
97
Sklar
L. A.
,
Omann
G. M.
,
Painter
R. G.
(
1985
).
Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular Ca++.
J. Cell Biol
101
,
1161
1166
Stoyanov
B.
,
Volinia
S.
,
Hanck
T.
,
Rubio
I.
,
Loubtchenkov
M.
,
Malek
D.
,
Stoyanova
S.
,
Vanhaesebroeck
B.
,
Dhand
R.
,
Nurnberg
B.
, et al. 
(
1995
).
Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.
Science
269
,
690
693
Traynor-Kaplan
A. E.
,
Thompson
B. L.
,
Harris
A. L.
,
Taylor
P.
,
Omann
G. M.
,
Sklar
L. A.
(
1989
).
Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils.
J. Biol. Chem
264
,
15668
15673
Vogel
G.
(
1987
).
Endocytosis and recognition mechanisms in Dictyostelium discoideum.
Meth. Cell Biol
28
,
129
137
Wennstrom
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 beta-receptor require the binding site for phosphatidylinositol 3kinase.
Oncogene
9
,
651
660
Yamauchi
K.
,
Holt
K.
,
Pessin
J. E.
(
1993
).
Phosphatidylinositol 3-kinase functions upstream of Ras and Raf in mediating insulin stimulation of c-fos transcription.
J. Biol. Chem
268
,
14597
14600
Yao
J.
,
Harvath
L.
,
Gilbert
D. L.
,
Colton
C. A.
(
1990
).
Chemotaxis by a CNS macrophage, the microglia.
J. Neurosci. Res
27
,
36
42
Zheng
Y.
,
Bagrodia
S.
,
Cerione
R. A.
(
1994
).
Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85.
J. Biol. Chem
269
,
18727
18730
Zhou
K.
,
Takegawa
K.
,
Emr
S. D.
,
Firtel
R. A.
(
1995
).
A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.
Mol. Cell. Biol
15
,
5645
5656
This content is only available via PDF.