{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T07:29:43Z","timestamp":1792654183866,"version":"4.5.0"},"reference-count":53,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5492","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,10,27]]},"abstract":"<jats:p>\n            The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2\/3 (Arp2\/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP\n            <jats:sub>2<\/jats:sub>\n            ) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP\n            <jats:sub>2<\/jats:sub>\n            -binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2\/3 complex in an inactive \u201cclosed\u201d conformation. In this state, both the Cdc42- and PIP\n            <jats:sub>2<\/jats:sub>\n            -binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.\n          <\/jats:p>","DOI":"10.1126\/science.290.5492.801","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:47:44Z","timestamp":1027763264000},"page":"801-806","source":"Crossref","is-referenced-by-count":454,"title":["Integration of Multiple Signals Through Cooperative Regulation of the N-WASP-Arp2\/3 Complex"],"prefix":"10.1126","volume":"290","author":[{"given":"Kenneth E.","family":"Prehoda","sequence":"first","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology, and"},{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143\u20130450, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica A.","family":"Scott","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology, and"},{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143\u20130450, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. Dyche","family":"Mullins","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology, and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wendell A.","family":"Lim","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology, and"},{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143\u20130450, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.284.5415.765"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81281-7"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1074-5521(99)80107-0"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(99)00061-7"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80732-1"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)00015-3"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80696-0"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.279.5350.509"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/35004513"},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/34208"},{"key":"e_1_3_1_12_2","unstructured":"The specific sequence of the basic motif [NISHTKEKKKGKAKKKRLTK (24)] is essential for repression; mutation of nine lysines to alanine abolishes repression (K. E. Prehoda W. A. Lim data not shown)."},{"key":"e_1_3_1_13_2","unstructured":"The measured affinity for the WASP GBD-VCA interaction is K d \u2248 1 \u03bcM (9)."},{"key":"e_1_3_1_14_2","unstructured":"K. E. Prehoda W. A. Lim data not shown."},{"key":"e_1_3_1_15_2","unstructured":"Like full-length N-WASP the degree of mini\u2013N-WASP activation is dependent on the nucleotide state of Cdc42 (5). Our findings with mini\u2013N-WASP show that the enabled\/VASP homology 1 (EVH1) domain and proline-rich region are unnecessary for this mode of regulation. These other domains may be involved in targeting ["},{"key":"e_1_3_1_15_3","doi-asserted-by":"publisher","DOI":"10.1038\/35017080"},{"key":"e_1_3_1_15_4","unstructured":"] or in modes of regulation distinct from that mediated by the control region. Our findings contradict previous suggestions that the EVH1 domain is the input point for PIP 2 activation (5) ["},{"key":"e_1_3_1_15_5","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1996.tb00917.x"},{"key":"e_1_3_1_15_6","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80757-6"},{"key":"e_1_3_1_15_7","unstructured":"]. We found that the N-WASP EVH1 domain is a peptide-binding module and does not bind acidic phospholipids (K. E. Prehoda J. A. Scott W. A. Lim in preparation)."},{"key":"e_1_3_1_16_2","unstructured":"Addition of high concentrations of the control region can apparently inhibit interaction of Arp2\/3 with the VCA domain. However interpretation of this experiment is complicated by our subsequent finding that there is a second Arp2\/3-binding site in the control region (see below) which can at stoichiometric excess competitively deplete available Arp2\/3."},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80792-8"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.112.23.4175"},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(63)80091-1"},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","unstructured":"M. F. Perutz Sci. Am. 239 92 (December 1978).","DOI":"10.1038\/scientificamerican1278-92"},{"key":"e_1_3_1_21_2","unstructured":"This value is likely to be a lower estimate for the cooperativity in vivo since activated Cdc42 is normally prenylated ["},{"key":"e_1_3_1_21_3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.65.070196.001325"},{"key":"e_1_3_1_21_4","unstructured":"]. Thus both inputs would be restricted to the membrane and their effective concentrations with respect to one another would be even higher."},{"key":"e_1_3_1_22_2","unstructured":"These results show that exclusion of Arp2\/3 is not required for repression. However it is possible that other elements in full-length N-WASP function by blocking Arp2\/3 binding."},{"key":"e_1_3_1_23_2","unstructured":"This model predicts that N-WASP will have a limited range of optimal sensitivity. High concentrations of either individual input can overload the system consistent with observations that either overexpression of a Cdc42 exchange factor ["},{"key":"e_1_3_1_23_3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.52.33169"},{"key":"e_1_3_1_23_4","unstructured":"] or phosphoinositide 5-kinase ["},{"key":"e_1_3_1_23_5","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(00)00384-5"},{"key":"e_1_3_1_23_6","unstructured":"] results in unregulated actin polymerization. N-WASP's signal integration properties are likely optimized for the physiologically relevant range of input concentrations just as the oxygen-binding properties of hemoglobin are set for the observed range of oxygen partial pressure [M. Perutz Cooperativity and Allosteric Regulation in Proteins (Cambridge Univ. Press Cambridge 1990)]."},{"key":"e_1_3_1_24_2","first-page":"65","volume":"72","author":"Bourne H. R.","year":"1993","unstructured":"Bourne H. R., Nicoll R., Cell (Suppl.) 72, 65 (1993).","journal-title":"Cell (Suppl.)"},{"key":"e_1_3_1_25_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_26_2","unstructured":"N-WASP fragments were cloned by polymerase chain reaction from a rat cDNA library. Proteins were expressed as fusions to either a polyhistidine tag or glutathione S -transferase (GST). Proteins were expressed in Escherichia coli (BL21-DE3) and purified by chromatography on Ni-NTA resin (Qiagen) or glutathione agarose resin (Sigma) followed by Source S or Q resins (Pharmacia). The polyhistidine tag was removed by incubation with tobacco etch virus (TEV) protease except when required for detection in Western blots."},{"key":"e_1_3_1_27_2","unstructured":"Actin polymerization assays were performed as described ["},{"key":"e_1_3_1_27_3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.7.3739"},{"key":"e_1_3_1_27_4","unstructured":"] . Briefly polymerization of actin doped with 1.2% pyrene-labeled actin was monitored via change in fluorescence (excitation: 366 nm; emission: 407 nm). Before the reaction actin was converted from the Ca-ATP to the Mg-ATP form by adding MgCl 2 and EGTA to final concentrations of 50 and 200 \u03bcM respectively and incubating at 25\u00b0C for 2 min. To initiate polymerization actin was added to a solution containing Arp2\/3 and any other factors at 20\u00b0C. Final conditions were 2.5 \u03bcM total actin 50 mM KCl 1 mM MgCl 2 1 mM EGTA 10 mM imidazole (pH 7.0) 0.2 mM ATP and 1 mM DTT in a volume of 100 \u03bcl. Arp2\/3 and actin were purified from Acanthamoeba castelanii as described in the above reference. Rabbit skeletal muscle actin was pyrene-labeled as described ["},{"key":"e_1_3_1_27_5","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(80)90619-4"},{"key":"e_1_3_1_28_2","unstructured":"GST-fusion binding assays were performed by immobilizing GST-fusion proteins on glutathione agarose beads (Sigma). We incubated 5 \u03bcl of beads with the appropriate target proteins in binding buffer [20 mM HEPES (pH 7.0) 100 mM NaCl 1 mM dithiothreitol (DTT)] in a total volume of 100 \u03bcl (final GST-fusion concentration of \u223c10 \u03bcM). After gentle rotation at 4\u00b0C for 30 min beads were washed three times with 1 ml binding buffer plus 0.05% Tween-20. Bound proteins were analyzed by SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by coomassie blue staining or by Western blotting with anti-Arp2 or anti-polyhistidine antibodies (Qiagen)."},{"key":"e_1_3_1_29_2","unstructured":"Human Cdc42 was expressed in E. coli as a fusion to GST and activated by incubating with 100 \u03bcM GTP\u03b3S for 30 min at room temperature followed by addition of MgCl 2 to a 10 mM final concentration ["},{"key":"e_1_3_1_29_3","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.146.6.1319"},{"key":"e_1_3_1_29_4","unstructured":"]. The GST tag was removed by thrombin cleavage at 4\u00b0C for 2 hours followed by purification on a Source Q column. Vesicles containing phosphatidylinositol 4-phosphate (PIP) or PIP 2 (48:48:4 of dibromo-phosphatidyl choline:phosphatidyl serine: PIP or PIP 2 ) were prepared by dissolving phospholipids in chloroform drying under nitrogen resuspending in 20 mM HEPES (pH 7.0) 100 mM NaCl and 1 mM DTT and passaging through >10 freeze-thaw\/bath sonication cycles. Approximate concentrations of PIP 2 or PIP were calculated by dividing the total concentration by two since 50% of the total was assumed to be on the inside layer of the vesicles."},{"key":"e_1_3_1_30_2","unstructured":"Cosedimentation lipid vesicle binding assays were performed as described ["},{"key":"e_1_3_1_30_3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.46.30497"},{"key":"e_1_3_1_30_4","unstructured":"] by mixing 3 \u03bcM protein and vesicles (\u223c20 \u03bcM PIP or PIP 2 ) in 20 mM HEPES (pH 7.0) 100 mM NaCl and 1 mM DTT (total assay volume 65 \u03bcl) incubating at 25\u00b0C for 10 min and centrifuging at 100 000 g for 1 hour. The vesicle pellet was washed with 200 \u03bcl of buffer and suspended in a volume of buffer equal to the supernatant. Bound (pellet) and unbound proteins (supernatant) were identified by SDS-PAGE."},{"key":"e_1_3_1_31_2","unstructured":"Supplemental figures are available at www.sciencemag.org\/feature\/data\/1054208.shl"},{"key":"e_1_3_1_32_2","unstructured":"Although we found that repressed mini\u2013N-WASP still interacts with Arp2\/3 we do not know whether Arp2\/3 binds to a composite site including elements from both the VCA and B-GBD fragments (as modeled in Fig. 2D) or if it only interacts with the B-GBD site. We only know that the second binding site helps shift the VCA-Arp2\/3 complex into a nonproductive conformation."},{"key":"e_1_3_1_33_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.29.18067"},{"key":"e_1_3_1_34_2","unstructured":"Simulation of cooperativity model. The fractional activity of N-WASP was calculated to be  \u03b8=KB[A]+KA[B]+c[A][B]KAKB+KB[A]+KA[B]+c[A][B]where [W] is the concentration of mini\u2013N-WASP [A] is the total concentration of input molecule A [B] is the total concentration of input molecule B c is the cooperativity factor linking binding of A and B and  KA=[W][A][W\u00b7A]\u00a0KB=[W][B][W\u00b7B] KAc=[W\u00b7B][A][W\u00b7A\u00b7B]\u00a0KBc=[W\u00b7A][B][W\u00b7A\u00b7B]This model assumes that [W tot ] << [A] and [B] and that species WA WB and WAB have equal activity (W is inactive). Theoretical curves describing the behavior for different values of c were calculated using the program Mathematica (Wolfram Research www.wolfram.com)."},{"key":"e_1_3_1_35_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.150.6.1299"},{"key":"e_1_3_1_36_2","unstructured":"We thank J. Zalevsky for reagents and helpful discussions and H. Bourne V. Denic D. Julius B. Kelch H. Madhani J. Weissman and members of the Lim lab for comments and discussion. Supported by grants to W.A.L. from NIH the Burroughs Wellcome Young Investigator Program the Searle Scholars Program and the Packard Foundation. K.E.P. is a Cancer Research Institute Postdoctoral Fellow."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.290.5492.801","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T10:43:53Z","timestamp":1705142633000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.290.5492.801"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,10,27]]},"references-count":53,"journal-issue":{"issue":"5492","published-print":{"date-parts":[[2000,10,27]]}},"alternative-id":["10.1126\/science.290.5492.801"],"URL":"https:\/\/doi.org\/10.1126\/science.290.5492.801","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,10,27]]}}}