Interaction of cortactin and N-WASp with Arp2/3 complex.
BACKGROUND
Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding.


RESULTS
We investigated how cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of cortactin to Arp2/3 complex.


CONCLUSIONS
Under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.
Weaver
Alissa M
AM
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Heuser
John E
JE
Karginov
Andrei V
AV
Lee
Wei-lih
WL
Parsons
J Thomas
JT
Cooper
John A
JA
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D051356:Cortactin; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013950:Thymus Gland / Q000378:metabolism; D014364:Tryptophan; D014923:Wiskott-Aldrich Syndrome; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D051356:Cortactin; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D014364:Tryptophan