For a detailed analysis of the profilin-actin interaction, we designed several point mutations in bovine profilin I by computer modeling. The recombinant proteins were analyzed in vitro for their actin-binding properties. Mutant proteins with a putatively higher affinity for actin were produced by attempting to introduce an additional bond to actin. However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting that additional putative bonds created this way cannot increase profilin's affinity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired properties in viscosity assays, while their binding sites for poly(L)proline were still intact. The profilin mutant F59A, with an affinity for actin reduced by one order of magnitude as compared to wild-type profilin, was analyzed further in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profilins are targeted to and tethered at these sites by ligands other than actin. Profilin null cells of Dictyostelium were transfected with bovine wild-type profilin I and F59A. Bovine profilin I, although expressed to only approximately 10% of the endogenous profilin level determined for wild-type Dictyostelium, caused a substantial rescue of the defects observed in profilin null amoebae, as seen by measuring the growth of colony surface areas and the percentage of polynucleated cells. The mutant protein was much less effective. These results emphasize the highly conserved biological function of profilins with low sequence homology, and correlate specifically their actin-binding capacity with cell motility and proliferation.

REFERENCES

Aderem
A.
(
1992
).
Signal transduction and the actin cytoskeleton: the roles of MARCKS and profilin.
Trends Biochem. Sci
17
,
438
443
Archer
S. J.
,
Vinson
V. K.
,
Pollard
T. D.
,
Torchia
D. A.
(
1994
).
Elucidation of the poly-L-proline binding site in Acanthamoeba profilin I by NMR spectroscopy.
FEBS Lett
337
,
145
151
Balasubramanian
M. K.
,
Hirani
B. R.
,
Burke
J. D.
,
Gould
K. L.
(
1994
).
The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis.
J. Cell Biol
125
,
1289
1301
Ballweber
E.
,
Giehl
K.
,
Hannappel
E.
,
Huff
T.
,
Jockusch
B. M.
,
Mannherz
H. G.
(
1998
).
Plant profilin induces actin polymerization from actin: beta-thymosin complexes and competes directly with beta-thymosins and with negative co-operativity with DNase I for binding to actin.
FEBS Lett
425
,
251
255
Bjorkegren
C.
,
Rozycki
M.
,
Schutt
C. E.
,
Lindberg
U.
,
Karlsson
R.
(
1993
).
Mutagenesis of human profilin locates its poly(L-proline)-binding site to a hydrophobic patch of aromatic amino acids.
FEBS Lett
333
,
123
126
Bjorkegren-Sjogren
C.
,
Korenbaum
E.
,
Nordberg
P.
,
Lindberg
U.
,
Karlsson
R.
(
1997
).
Isolation and characterization of two mutants of human profilin I that do not bind poly(L-proline).
FEBS Lett
418
,
258
264
Buss
F.
,
Jockusch
B. M.
(
1989
).
Tissue-specific expression of profilin.
FEBS Lett
249
,
31
34
Buss
F.
,
Temm-Grove
C.
,
Henning
S.
,
Jockusch
B. M.
(
1992
).
Distribution of profilin in fibroblasts correlates with the presence of highly dynamic actin filaments.
Cell Motil. Cytoskel
22
,
51
61
Cao
L. G.
,
Babcock
G. G.
,
Rubenstein
P. A.
,
Wang
Y. L.
(
1992
).
Effects of profilin and profilactin on actin structure and function in living cells.
J. Cell Biol
117
,
1023
1029
Carlsson
L.
,
Nystrom
L. E.
,
Sundkvist
I.
,
Markey
F.
,
Lindberg
U.
(
1977
).
Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells.
J. Mol. Biol
115
,
465
483
Cooley
L.
,
Verheyen
E.
,
Ayers
K.
(
1992
).
chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.
Cell
69
,
173
184
Dunn
J. J.
,
Studier
F. W.
(
1983
).
Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements.
J. Mol. Biol
166
,
477
535
Finkel
T.
,
Theriot
J. A.
,
Dise
K. R.
,
Tomaselli
G. F.
,
Goldschmidt-Clermont
P. J.
(
1994
).
Dynamic actin structures stabilized by profilin.
Proc. Nat. Acad. Sci. USA
91
,
1510
1514
Gertler
F. B.
,
Niebuhr
K.
,
Reinhard
M.
,
Wehland
J.
,
Soriano
P.
(
1996
).
Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell
87
,
227
239
Giehl
K.
,
Valenta
R.
,
Rothkegel
M.
,
Ronsiek
M.
,
Mannherz
H. G.
,
Jockusch
B. M.
(
1994
).
Interaction of plant profilin with mammalian actin.
Eur. J. Biochem
226
,
681
689
Goldschmidt-Clermont
P. J.
,
Kim
J. W.
,
Machesky
L. M.
,
Rhee
S. G.
,
Pollard
T. D.
(
1991
).
Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.
Science
251
,
1231
1233
Goldschmidt-Clermont
P. J.
,
Machesky
L. M.
,
Baldassare
J. J.
,
Pollard
T. D.
(
1990
).
The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C.
Science
247
,
1575
1578
Goldschmidt-Clermont
P. J.
,
Machesky
L. M.
,
Doberstein
S. K.
,
Pollard
T. D.
(
1991
).
Mechanism of the interaction of human platelet profilin with actin.
J. Cell Biol
113
,
1081
1089
Haarer
B. K.
,
Lillie
S. H.
,
Adams
A. E.
,
Magdolen
V.
,
Bandlow
W.
,
Brown
S. S.
(
1990
).
Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.
J. Cell Biol
110
,
105
114
Haarer
B. K.
,
Petzold
A. S.
,
Brown
S. S.
(
1993
).
Mutational analysis of yeast profilin.
Mol. Cell. Biol
13
,
7864
7873
Hajkova
L.
,
Bjorkegren Sjogren
C.
,
Korenbaum
E.
,
Nordberg
P.
,
Karlsson
R.
(
1997
).
Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo.
Exp. Cell Res
234
,
66
77
Hartwig
J. H.
,
Chambers
K. A.
,
Hopcia
K. L.
,
Kwiatkowski
D. J.
(
1989
).
Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.
J. Cell Biol
109
,
1571
1579
Haugwitz
M.
,
Noegel
A. A.
,
Karakesisoglou
J.
,
Schleicher
M.
(
1994
).
Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development.
Cell
79
,
303
314
Haugwitz
M.
,
Noegel
A. A.
,
Rieger
D.
,
Lottspeich
F.
,
Schleicher
M.
(
1991
).
Dictyostelium discoideum contains two profilin isoforms that differ in structure and function.
J. Cell Sci
100
,
481
489
Houk
T. W.
Jr.
,
Ue
K.
(
1974
).
The measurement of actin concentration in solution: a comparison of methods.
Anal. Biochem
62
,
66
74
Kaiser
D. A.
,
Goldschmidt-Clermont
P. J.
,
Levine
B. A.
,
Pollard
T. D.
(
1989
).
Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns.
Cell Motil. Cytoskel
14
,
251
262
Karakesisoglou
I.
,
Schleicher
M.
,
Gibbon
B. C.
,
Staiger
C. J.
(
1996
).
Plant profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium cells.
Cell Motil. Cytoskel
34
,
36
47
Korenbaum
E.
,
Nordberg
P.
,
Bjorkegren-Sjogren
C.
,
Schutt
C. E.
,
Lindberg
U.
,
Karlsson
R.
(
1998
).
The role of profilin in actin polymerization and nucleotide exchange.
Biochemistry
37
,
9274
9283
Kouyama
T.
,
Mihashi
K.
(
1981
).
Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin.
Eur. J. Biochem
114
,
33
38
Lange
K.
,
Brandt
U.
,
Gartzke
J.
,
Bergmann
J.
(
1998
).
Action of insulin on the surface morphology of hepatocytes: role of phosphatidylinositol 3-kinase in insulin-induced shape change of microvilli.
Exp. Cell Res
239
,
139
151
Larsson
H.
,
Lindberg
U.
(
1988
).
The effect of divalent cations on the interaction between calf spleen profilin and different actins.
Biochim. Biophys. Acta
953
,
95
105
Lassing
I.
,
Lindberg
U.
(
1985
).
Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin.
Nature
314
,
472
474
Lassing
I.
,
Lindberg
U.
(
1988
).
Specificity of the interaction between phosphatidylinositol 4,5-bisphosphate and the profilin:actin complex.
J. Cell Biochem
37
,
255
267
Lindberg
U.
,
Schutt
C. E.
,
Hellsten
E.
,
Tjader
A. C.
,
Hult
T.
(
1988
).
The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes.
Biochim. Biophys. Acta
967
,
391
400
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
Machesky
L. M.
,
Atkinson
S. J.
,
Ampe
C.
,
Vandekerckhove
J.
,
Pollard
T. D.
(
1994
).
Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.
J. Cell Biol
127
,
107
115
Mayboroda
O.
,
Schluter
K.
,
Jockusch
B. M.
(
1997
).
Differential colocalization of profilin with microfilaments in PtK2 cells.
Cell Motil. Cytoskel
37
,
166
177
Metzler
W. J.
,
Bell
A. J.
,
Ernst
E.
,
Lavoie
T. B.
,
Mueller
L.
(
1994
).
Identification of the poly-L-proline-binding site on human profilin.
J. Biol. Chem
269
,
4620
4625
Neujahr
R.
,
Albrecht
R.
,
Kohler
J.
,
Matzner
M.
,
Schwartz
J. M.
,
Westphal
M.
,
Gerisch
G.
(
1998
).
Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells.
J. Cell Sci
111
,
1227
1240
Nishida
E.
,
Maekawa
S.
,
Sakai
H.
(
1984
).
Characterization of the action of porcine brain profilin on actin polymerization.
J. Biochem
95
,
399
404
Nystrom
L. E.
,
Lindberg
U.
,
Kendrick-Jones
J.
,
Jakes
R.
(
1979
).
The amino acid sequence of profilin from calf spleen.
FEBS Lett
101
,
161
165
Ostrander
D. B.
,
Gorman
J. A.
,
Carman
G. M.
(
1995
).
Regulation of profilin localization in Saccharomyces cerevisiae by phosphoinositide metabolism.
J. Biol. Chem
270
,
27045
27050
Pantaloni
D.
,
Carlier
M. F.
(
1993
).
How profilin promotes actin filament assembly in the presence of thymosin beta 4.
Cell
75
,
1007
1014
Perelroizen
I.
,
Didry
D.
,
Christensen
H.
,
Chua
N. H.
,
Carlier
M. F.
(
1996
).
Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly.
J. Biol. Chem
271
,
12302
12309
Podolski
J. L.
,
Steck
T. L.
(
1990
).
Length distribution of F-actin in Dictyostelium discoideum.
J. Biol. Chem
265
,
1312
1318
Pollard
T. D.
,
Cooper
J. A.
(
1984
).
Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation.
Biochemistry
23
,
6631
6641
Pring
M.
,
Weber
A.
,
Bubb
M. R.
(
1992
).
Profilin-actin complexes directly elongate actin filaments at the barbed end.
Biochemistry
31
,
1827
1836
Reinhard
M.
,
Giehl
K.
,
Abel
K.
,
Haffner
C.
,
Jarchau
T.
,
Hoppe
V.
,
Jockusch
B. M.
,
Walter
U.
(
1995
).
The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.
EMBO J
14
,
1583
1589
Rothkegel
M.
,
Mayboroda
O.
,
Rohde
M.
,
Wucherpfennig
C.
,
Valenta
R.
,
Jockusch
B. M.
(
1996
).
Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells.
J. Cell Sci
109
,
83
90
Rozycki
M. D.
,
Myslik
J. C.
,
Schutt
C. E.
,
Lindberg
U.
(
1994
).
Structural aspects of actin-binding proteins.
Curr. Opin. Cell Biol
6
,
87
95
Schagger
H.
,
von Jagow
G.
(
1987
).
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Anal. Biochem
166
,
368
379
Schluter
K.
,
Jockusch
B. M.
,
Rothkegel
M.
(
1997
).
Profilins as regulators of actin dynamics.
Biochim. Biophys. Acta
1359
,
97
109
Schluter
K.
,
Zeuschner
D.
,
Winkler
J.
,
Pope
B.
,
Weeds
A.
,
Jockusch
B.
(
1998
).
An alpha-actinin-profilin chimaera with two alternatively operating actin binding sites.
Eur. J. Cell Biol
76
,
1
8
Schomburg
D.
,
Reichelt
J.
(
1988
).
BRAGI: A comprehensive protein modeling program system.
J. Mol. Graphics
6
,
161
165
Schutt
C. E.
,
Myslik
J. C.
,
Rozycki
M. D.
,
Goonesekere
N. C.
,
Lindberg
U.
(
1993
).
The structure of crystalline profilin-beta-actin.
Nature
365
,
810
816
Sharp
P. M.
,
Devine
K. M.
(
1989
).
Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ‘prefer’ optimal codons.
Nucl. Acids Res
17
,
5029
5039
Simpson
P. A.
,
Spudich
J. A.
,
Parham
P.
(
1984
).
Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin.
J. Cell Biol
99
,
287
295
Sohn
R. H.
,
Chen
J.
,
Koblan
K. S.
,
Bray
P. F.
,
Goldschmidt-Clermont
P. J.
(
1995
).
Localization of a binding site for phosphatidylinositol 4,5-bisphosphate on human profilin.
J. Biol. Chem
270
,
21114
21120
Sohn
R. H.
,
Goldschmidt-Clermont
P. J.
(
1994
).
Profilin: at the crossroads of signal transduction and the actin cytoskeleton.
BioEssays
16
,
465
472
Spudich
J. A.
,
Watt
S.
(
1971
).
The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin.
J. Biol. Chem
246
,
4866
4871
Sussman
M.
(
1987
).
Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions.
Methods Cell Biol
28
,
9
29
Sutoh
K.
(
1993
).
A transformation vector for Dictyostelium discoideum with a new selectable marker bsr.
Plasmid
30
,
150
154
Tanaka
M.
,
Shibata
H.
(
1985
).
Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin.
Eur. J. Biochem
151
,
291
297
Tilney
L. G.
,
Bonder
E. M.
,
Coluccio
L. M.
,
Mooseker
M. S.
(
1983
).
Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin.
J. Cell Biol
97
,
112
124
Tseng
P. C.
,
Pollard
T. D.
(
1982
).
Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2.
J. Cell Biol
94
,
213
218
Vandekerckhove
J. S.
,
Kaiser
D. A.
,
Pollard
T. D.
(
1989
).
Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin.
J. Cell Biol
109
,
619
626
Verheyen
E. M.
,
Cooley
L.
(
1994
).
Profilin mutations disrupt multiple actin-dependent processes during Drosophila development.
Development
120
,
717
728
Watanabe
N.
,
Madaule
P.
,
Reid
T.
,
Ishizaki
T.
,
Watanabe
G.
,
Kakizuka
A.
,
Saito
Y.
,
Nakao
K.
,
Jockusch
B. M.
,
Narumiya
S.
(
1997
).
p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin.
EMBO J
16
,
3044
3056
Williams
K. L.
,
Newell
P. C.
(
1976
).
A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis.
Genetics
82
,
287
307
Witke
W.
,
Podtelejnikov
A. V.
,
Di Nardo
A.
,
Sutherland
J. D.
,
Gurniak
C. B.
,
Dotti
C.
,
Mann
M.
(
1998
).
In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.
EMBO J
17
,
967
976
Witke
W.
,
Sharpe
A. H.
,
Kwiatkowski
D. J.
(
1993
).
Profilin deficient mice are not viable.
Mol. Biol. Cell
4
,
149
–.
This content is only available via PDF.