Profilin enhances Cdc42-induced nucleation of actin polymerization.
We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.
Yang
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C
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.
Huang
M
M
DeBiasio
J
J
Pring
M
M
Joyce
M
M
Miki
H
H
Takenawa
T
T
Zigmond
S H
SH
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003285:Contractile Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000502:physiology; D010455:Peptides / Q000378:metabolism; D051304:Profilins; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D003285:Contractile Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D010455:Peptides; D051304:Profilins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; C011083:polyproline; D020764:cdc42 GTP-Binding Protein