Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Apr;14(4):1624-37.
doi: 10.1091/mbc.e02-08-0494.

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis

Jennifer L Zamanian  1 , Regis B Kelly
Affiliations

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis

Jennifer L Zamanian et al. Mol Biol Cell. 2003 Apr.

Abstract

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The DHPH domains of intersectin 1L localize to actin ruffles. The DHPH domains of intersectin 1L are overexpressed in Cos cells and visualized with antibody no. 4199, anti-intersectin DH domain, and FITC-conjugated secondary antibody (A, C, and E). Filamentous actin is visualized with rhodamine-phalloidin (B, D, and F). Although much of the DHPH expression is cytosolic, there is colocalization with actin ruffles at the periphery (arrows in A and B) and on the dorsal surface (arrows in C and D) of the cells. (E) The anti-intersectin DH domain antibody has little background staining in vector-transfected Cos cells at the same photographic exposure.
Figure 2
Figure 2
The intersectin 1L DH domain can associate with Cdc42 and Rac1, but its exchange activity is focused on Cdc42. (A) Diagram of the intersectin 1L domain structure. The domain structure and aa position of the recombinant fusion proteins used in this study are displayed. (B) GST pull-downs of small GTPases by the DH domain from rat brain cytosol. GST, GST DH, and GST DHPH, 6 μM, in 5 mg rat brain cytosol in binding buffer. GST alone does not pull-down any small GTPase. GST fusion proteins containing the DH domain pull-down Rac1 and Cdc42, but not RhoA. Overexposure of the Cdc42 samples allows normalization to the starting material and reveals that Cdc42 recovery is significantly higher than that of Rac1. (C) In vitro exchange reaction time course. GST and GST DHPH fusion protein, 750 nM, were assayed for exchange activity using 250 nM GST RhoA, GST Rac1, and GST Cdc42. [35S]GTPγS loading by GST on GST RhoA (□), GST DHPH on GST RhoA (▪), GST on GST Rac1 (○), GST DHPH on GST Rac1 (●), GST on GST Cdc42 (▵), and GST DHPH on GST Cdc42 (▴) as shown. The intersectin 1L DH domain exchange activity is specific for Cdc42. (D) GST pull-downs of intersectin L from rat brain cytosol by GST small GTPases. GST, GST RhoA, GST Rac1, and GST Cdc42, 2.3 μM, were rotated overnight in 5 mg rat brain cytosol at 4°C in binding buffer. After elution from glutathione agarose, the samples were blotted with antibody no. 4396 that recognizes both the long and short forms of intersectin 1. GST Cdc42 pulls down intersectin L, but not intersectin S. GST RhoA and GST Rac1 do not pull down any intersectin.
Figure 3
Figure 3
The upstream SH3 domains, but not the downstream PH and C2 domains, inhibit the DH domain exchange activity in vitro. Incorporation of [35S]GTPγS into Cdc42 was measured. (A) In vitro exchange reaction kinetics were measured in experiments using 50 or 75 nM of GST (□), GST DHPH (○), or GST SH3ABCDE-DHPH (▵) and 500 nM recombinant GST Cdc42. The SH3ABCDE domains added in cis with the DHPH domains inhibit the exchange activity of the DH domain. (B) The exchange index at 10 min is a comparison of the exchange activity of the DHPH domains with the SH3ABCDE, SH3CDE, and SH3E domains added in cis relative to the exchange activity of the DHPH domains alone. The GST background cpm have been subtracted out (see MATERIALS AND METHODS). The single SH3E domain inhibits (50%) the DH exchange activity, but additional SH3 domains may increase the inhibitory activity. (C) In vitro exchange reaction kinetics were measured using 75 nM GST (□), GST DH (○), or GST DHPH (▵) on 1 μM Cdc42. The exchange activity of the DH domain alone is reduced relative to the DHPH domain. (D) In vitro exchange reaction time course. Fusion proteins, 75 nM, were assayed for exchange activity on 1.25 μM Cdc42 in the presence or absence of 640 μM Ca2+. GST (□), GST + Ca2+ (▪), GST DHPH (○), GST DHPH + Ca2+ (●), GST DHPHC2 (▵), and GST DHPHC2 + Ca2+ (▴). The C2 domain added in cis does not strongly affect the DH domain activity in either the presence or the absence of Ca2+.
Figure 4
Figure 4
The SH3 domains directly inhibit the binding of Cdc42 to the DH domain. (A) GST pull-downs of Cdc42 from rat brain cytosol by GST DH domain fusion proteins. GST fusion proteins, 2.5 μM, containing the DH were incubated in 1.5 mg rat brain cytosol in binding buffer. Glutathione agarose beads were added to pull-down the protein after overnight incubation. Cdc42 is pulled down by all the DH domain-containing proteins. However, the SH3CDE domains in cis strongly inhibit the binding of Cdc42 to the DH domain. (B) Quantification of Cdc42 pull-down results. The Western blot in A was quantified using the NIH Image 1.61 program. The SH3CDE domains inhibit Cdc42 binding from rat brain cytosol by 80–95%. GST pull-down using recombinant Cdc42 instead of cytosol. (C) GST DH domain containing fusion proteins, 10 nM, and 160 nM Cdc42 were incubated in binding buffer + 0.1% Triton X-100. Recombinant Cdc42 bound to all the DH domain-containing fusion proteins. The SH3CDE domains again inhibit binding of Cdc42 to the DHPH domains. (D) Cdc42 binding was normalized to GST fusion protein immunoreactivity using NIH Image 1.6 program. The ratio of Cdc42 binding to GST fusion protein is expressed in arbitrary units. Binding of Cdc42 to the DHPH domains is inhibited 70% by the SH3CDE domains. Binding of Cdc42 to the DH domain alone is also 70% lower than to the DHPH domains. No further reduction in Cdc42 binding to the DH domain alone is seen by addition of the SH3CDE domains.
Figure 5
Figure 5
Evidence for an intramolecular interaction between the intersectin 1L SH3 and DH domains. (A) In vitro exchange reaction kinetics were measured using 250 nM GST (□), GST DH (○), or GST SH3CDE-DH (▵) on 533 nM GST Cdc42. The exchange activity of the DH domain in the absence of the PH domain is reduced by the addition of the SH3CDE domains in cis. (B) GST pull-downs of intersectin 1 from rat brain cytosol by the intersectin 1L SH3 domains. GST SH3, 5 μM, containing fusion in 1.5 mg rat brain cytosol in MgCl2 binding buffer. Western blotting of glutathione agarose eluates was done with antibody no. 4396. The GST SH3ABCDE fusion protein binds intersectin 1L protein. The SH3ABCDE-DHPH fusion protein pulls down larger amounts of intersectin 1L. (C) Time course of an in vitro exchange reaction, where SH3 domains are added in trans. Incorporation of [35S]GTPγS into Cdc42 was followed. Data compiled from several experiments using 25 nM GST + 10–20 μM GST (□), 25 nM GST DHPH + 10–20 μM GST (○), or 25 nM GST DHPH + 10–20 μM (depending on the experiment) recombinant SH3ABCDE in trans (▵) were assayed for exchange activity on 1.25 μM Cdc42. The SH3ABCDE domains added in trans inhibit the activity of the DHPH domains using pure recombinant proteins. (D) The exchange index at 10 min compares fusion protein exchange activity to that of the DHPH domains. The SH3ABCDE domains in trans completely inhibited the exchange activity of the DHPH domains. (E) The SH3 domains bind the DH domain. Binding of recombinant 6× His DH (5 μM) and 6× His DHPH (5 μM) domains by individual and tandem GST SH3 domains (15 μM) in MgCl2 binding buffer. Western blotting of glutathione agarose eluates was done with anti-6× His Tag antibody. The individual SH3A, C, and D domains and the SH3ABCDE domains in tandem bind the DH domain. The SH3E domain binds to the DHPH domains.
Figure 6
Figure 6
The SH3 domains do not interact with and inhibit the DH domain through their PxxP binding pocket. (A) Inhibition of SH3 domain pull-down of N-WASP from rat brain cytosol by PxxP binding pocket mutations. 5 μM GST fusion proteins in 1.5 mg rat brain cytosol in MgCl2 binding buffer. The GST SH3E-DHPH and GST SH3DE-DPH fusion proteins pull down N-WASP. The binding pocket mutations in the SH3D and SH3E domains abolish binding to N-WASP. Note that the GST SH3E DHPH fusion protein distorts the N-WASP band by running on top of it. (B) Time course of an in vitro exchange reaction with the SH3 mutants. GST (□), GST DHPH (▿), GST SH3E-DHPH (▵) and GST SH3E (P1198L)-DHPH (○), 75 nM, were assayed for exchange activity on 1.25 μM Cdc42. (C) The exchange index at 10 min compares fusion protein exchange activity to that of the DHPH domains. The GST SH3E-DHPH and GST SH3DE-DHPH fusion protein exchange activities are inhibited 50% relative to GST DHPH alone. The P1122L and P1198L mutations in the SH3D and SH3E domains, respectively, that abolish binding to N-WASP do not effect this inhibition.
Figure 7
Figure 7
Binding of protein to the SH3 domains does not relieve the inhibition of the DH domain in vitro. (A) GST pull-down of recombinant dynamin with DH domain-containing fusion proteins is independent of GTP. GST fusion proteins, 50 nM, containing the DH domain in MgCl2 binding buffer + 1 μM BSA or 1 μM dynamin rotated 4 h at 4°C. Approximately half of the GST SH3ABCDE-DHPH protein is bound by dynamin as quantified by Western blotting signal relative to known dynamin using NIH Image 1.61. The addition of guanine nucleotides or the C2 domain had no effect on dynamin binding. (B) Time course of in vitro exchange reactions with dynamin. The following were assayed for exchange activity on 750 nM GST Cdc42 after the loss of [3H]GDP from Cdc42: 75 nM GST + 2.5 μM BSA (□), 75 nM GST + 1.5 μM dynamin (▪), GST DHPH 75 nM + 2.5 μM BSA (○), 75 nM GST DHPH + 1.5 μM dynamin (●), 75 M GST SH3ABCDE-DHPH + 2.5 μM BSA (▵) and 75 nM GST SH3ABCDE-DHPH + 1.5 μM dynamin (▴) . Addition of dynamin to the exchange reaction does not effect the inhibition by the SH3 domains. (C) GST pull-downs of recombinant N-WASP with DH domain-containing fusion proteins. GST, GST DHPH, and GST SH3ABCDE-DHPH, 50 nM, in MgCl2 binding buffer + 0.5 mg/ml BSA and 450 nM N-WASP was rotated at 4°C for 4 h. Under these conditions approximately half of the GST SH3ABCDE-DHPH is bound to N-WASP as quantified by comparing the Western blotting signal to known N-WASP using NIH Image 1.61. (D) Time course of in vitro exchange reactions with N-WASP. The following were assayed for exchange of cold GTP on 1.25 μM Cdc42 for [3H]GDP: 75 nM GST + BSA (□), GST + 4.5 μM N-WASP (▪), GST DHPH + BSA (○), GST DHPH + 4.5 μM N-WASP (●), GST SH3ABCDE-DHPH + BSA (▵), and GST SH3ABCDE-DHPH + 4.5 μM N-WASP (▴). Addition of N-WASP to the exchange reaction does not effect the inhibition by the SH3 domains.
Figure 8
Figure 8
Dynamin and N-WASP compete for binding to the intersectin 1L SH3 domains. (A) GST pull-downs of dynamin and N-WASP from rat brain cytosol by individual SH3 domains. GST SH3 domains, 5 μM, in 1.5 mg rat brain cytosol in MgCl2 binding buffer. Dynamin binds to the SH3A, SH3C, and SH3E domains. N-WASP also binds to these domains, but also has some binding to SH3B and SH3D. (B) Dynamin competes with N-WASP for binding to the SH3 domains. 75 nM GST SH3ABCDE-DHPH in MgCl2 binding buffer. In lanes 1–4, 300 nM N-WASP binding in the presence of 55 nM, 113 nM, 750 nM, and 4.5 μM dynamin. In lanes 5–8, 450 nM dynamin binding in the presence of 37.5 nM, 75 nM, 500 nM, and 3 μM N-WASP. Lane 9 contains 3.75 pmol N-WASP. Lane 10 contains 5.6 pmol dynamin protein. When equimolar dynamin and N-WASP are present, lane 7, 450 nM dynamin and 500 nM N-WASP, dynamin preferentially binds. Increasing amounts of dynamin compete with N-WASP binding to the SH3 domains. Only at very high concentrations does N-WASP compete with dynamin for binding to the SH3 domains.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aghazadeh B, Lowry WE, Huang XY, Rosen MK. Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell. 2000;102:625–633. - PubMed
    1. Bustelo XR. Regulation of Vav proteins by intramolecular events. Front Biosci. 2002;7:d24–d30. - PubMed
    1. Cicchetti P, Mayer BJ, Thiel G, Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992;257:803–806. - PubMed
    1. Clark SG, Stern MJ, Horvitz HR. C. elegans cell-signaling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992;356:340–344. - PubMed
    1. Clift-O'Grady L, Desnos C, Lichtenstein Y, Faundez V, Horng JT, Kelly RB. Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes. Methods. 1998;16:150–159. - PubMed

Publication types

MeSH terms

LinkOut - more resources