The GTPase dynamin binds to and is activated by a subset of SH3 domains

Cell, Vol. 75, 25-36, October 8, 1993, Copyright © 1993 by Cell Press

The GTPase Dynamin Binds to and Is Activated by a Subset of SH3 Domains

Ivan Gout,* Ritu Dhand,* lan D. Hiles,* Michael J. Fry,* George Panayotou,* Pamela Das,* Oanh Truong,* Nicholas F. Totty,* Justin Hsuan,* Grant W. Booker,t lain D. Campbell,t and Michael D. Waterfield*$ *Ludwig Institute for Cancer Research London WlP 8BT England tDepartment of Biochemistry University of Oxford Oxford OX1 3QU England SDepartment of Biochemistry and Molecular Biology University College London WC1E 6BT England

Summary

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the in- teractive regulation of GTP-binding proteins.

Introduction

The activation of receptors with intrinsic or associated protein-tyrosine kinase activity triggers the assembly of signal recognition complexes that regulate second messen- ger generation and determine the fate of the activated receptor (UIIrich and Schlessinger, 1990). A primary role of the protein-tyrosine kinase, as we currently understand it, is to generate tyrosine-phosphorylated recognition motifs for the binding of proteins that c6ntain Src homology 2 (SH2) domains that are consequently recruited into signal transduction complexes (Koch et al., 1991). These bound proteins possessing SH2 domains may have intrinsic en- zymatic or regulatory activities, for example, phospholipase Cy (PLCy) or the GTPase-activating protein (GAP) for ras. Alternatively, they may serve purely as linker molecules to bring other enzymes or regulatory proteins into the signaling complex (Cantley et al., 1991). This latter group includes p85, which binds the catalytic p110 subunit of the phosphatidylinositol (PI) 3-kinase (Otsu et al., 1991;

Hiles et al., 1992), and the Grb2 protein (growth factor receptor-bound protein 2; also known as Drk, ASH, or Sem-5), which links protein-tyrosine kinase receptors, such as the epidermal growth factor (EGF) receptor, to the guanine n ucleotide exchange protein, Sos (Son of sevenless), and thus to the ras activation pathway (Clark et el., 1992; Lowenstein et al., 1992; Matuoka et el., 1992; Buday and Downward, 1993; Olivier et al., 1993; Simon et al., 1993).

The structural basis for SH2 domain-phosphotyrosine binding has been advanced by studies employing mutagenesis of the tyrosine phosphorylation sites in receptors and by the generation of synthetic phosphopeptides span- ning putative binding sites (which have been used in analysis of SH2 domain binding) using either recombinant domains or intact proteins (Anderson et al., 1990; Fantl et al., 1992; Kashishian et al., 1992; Pawson and Gish, 1992). The emerging picture suggests that individual protein- Iyrosine kinase receptors have multiple distinct SH2 domain-binding motifs that, upon phosphorylation, trigger interactions with specific SH2 domain-containing proteins involved in signal transduction complexes. The range of these proteins with which a particular receptor interacts may therefore provide a basis for the specificity of action of different protein-tyrosine kinase receptors in vivo.

The assembly of these signaling complexes may not, however, be entirely dependent on SH2 domain interactions alone. Many proteins with SH2 domains also contain one or more SH3 domains (Koch et al., 1991). The 50-60 amino acid SH3 domain has been found in a wide variety of proteins in addition to those known to be involved in protein-tyrosine kinase-linked functions (reviewed by Musac- chio et al., 1992a). Initial suggestions for SH3 domain function favored a role in mediating interactions with the cytoskeleton, partly because these domains were found in several cytoskeletal or cytoskeleton-associated proteins, including spectrin, myosin 1, and a yeast actin-binding protein, but also because mutagenesis studies with the v-Src and v-Fps transforming proteins supported such a role (Drubin et al., 1990; Koch et al., 1991). Progress toward the definition of binding motifs for SH3 domains and their function has been made by using glutathione S-trans- ferase (GST)-SH3 domain fusion proteins to screen expression libraries. Thus, cDNAs for a number of potential SH3 domain-binding proteins have been isolated. The first such protein described was 3BP1 (Cicchetti et al., 1992), which bound to the SH3 domain of the Abl protein- tyrosine kinase via a proline-rich sequence. Within the partial 3BP1 cDNA sequence, there was also a region of sequence similarity to the product of the breakpoint cluster region gene, Bcro This Bcr-related domain has subsequently been identified in a number of otherwise unrelated proteins and seems to define a novel GAP domain for specific small G proteins (reviewed by Fry, 1992). From these data, it has been predicted that SH3 domains might be involved in the control of small, ras-like G proteins (Paw- son and Gish, 1992; Mayer and Baltimore, 1993). The ear-

Cell 26

lier link to the cytoskeleton and membrane may therefore still prove to be relevant, as two small G proteins, p21 ~'c and p21 rh°, have recently been implicated in controlling membrane ruffling and formation of stress fibers in response to growth factors (Ridley and Hall, 1992; Ridley et al., 1992).

The solution and crystal structures of the SH3 domains of spectrin, pp60 ~-~c, PLC7, and p85a were recently presented (Musacchio et al., 1992b; Yu et al., 1992; Booker et al., 1993; Kohda et al., 1993; Koyama et al., 1993): all of these domains show a similar basic fold of a compact 13 barrel structure involving five antiparallel I~ strands. Solu- tion studies have also demonstrated the binding of proline- rich peptides to these structures and indicated a hydrophobic binding site on the SH3 domain that is lined with conserved aromatic residues and flanked by two charged loops (Yu et al., 1992; Booker et al., 1993). The structural studies were complemented by Ran et al. (1993), who have defined by mutagenesis a potential consensus binding motif in the 3BP1 protein (XPXXPPPZXP, where X is any amino acid and Z is a hydrophobic amino acid) for the Abl SH3 domain. In particular, the proline residues at positions 2, 7, and 10 in this motif were found to be essential for binding. The proline residues may therefore function to provide some conformational rigidity to this binding motif, while the surrounding charged residues supply the specificity required for sequence recognition by individual SH3 domains.

Finally, recent studies of Grb2, an adaptor protein composed entirely of two SH3 domains flanking an SH2 domain, have strengthened the link between SH3 domains and the regulation of small G protein function (Lowenstein et al., 1992). It had previously been demonstrated that the Caenorhabditis elegans homoiog of Grb2, Sem-5, linked an EGF receptor-like molecule to ras-like proteins in the signal transduction pathway leading to C. elegans vulval induction (Clark et al., 1992). Point mutations within the SH3 domains were shown to impair Sem-5 function. Lo- wenstein et al. (1992) went on to show that comicroinjec- tion of Grb2 with Ha-ras led to DNA synthesis in rat fibroblasts, whereas alone neither protein had any stimulatory effect. Grb2 was subsequently shown to bind to the EGF receptor via its SH2 domain in a ligand-dependent manner (Rozakis-Adcock et al., 1992). The latest observation has been that the Grb2 homolog from both the Drosophila and murine systems couples a receptor protein-tyrosine kinase to an activator of the ras guanine nucleotide exchange protein, Sos (Buday and Downward, 1993; Olivier et al., 1993; Simon et al., 1993). The Drosophila Grb2 homolog, Drk, has been shown-to bind to the C-terminal tail of the Sos protein in vitro (Olivier et al., 1993; Simon et al., 1993), while in murine fibroblasts, Grb2 coimmuno- precipitates with murine Sos (mSosl) in an EGF- dependent manner (Buday and Downward, 1993). Ap- propriate proline-rich sequences are found within the sequence of the Sos proteins that may mediate the binding of Grb2 via its SH3 domains (Olivier et al., 1993). Together, these results strongly suggest that Grb2 interacts with activated receptors via its SH2 domains and with the Sos guanine nucleotide exchange protein via one or both of

its SH3 domains. Interaction of Grb2-bound Sos with ras presumably leads to ras activation. Thus, at least one of the pathways between receptor protein-tyrosine kinases and ras would appear to have been essentially defined.

The modular structure of the noncatalytic p85 subunit of the PI 3-kinase has facilitated both structural and functional studies on this protein and has led to rapid advances in our understanding of its role in the heterodimeric PI 3-kinase complex (Panayotou and Waterfield, 1992). Yet despite these advances, the precise function of the SH3 domain in this adaptor molecule has remained elusive. In an attempt to address this question, we prepared GST- SH3 fusion proteins containing the SH3 domain from p85~t of the PI 3-kinase and 14 other SH3 domains. These recombinant SH3 domains were then used as affinity matrices to screen a bovine brain extract for proteins to which they could specifically bind. Several proteins were identified this way, including a known GTP-binding protein, dynamin, and we describe here the binding and functional aspects of its interactions with a panel of recombinant SH3 domains.

Results

Affinity Purification of SH3 Domain-Binding Proteins To study the function of SH3 domains, we used affinity methods to identify potential SH3 domain-binding proteins. An affinity matrix was prepared by immobilization of a GST-p85~z-SH3 domain fusion protein (GST-p85~t- SH3) on glutathione-Sepharose beads. This matrix was then used to probe 0.02% and 1.0% Triton X-100 extracts of bovine brain for putative SH3 domain-binding proteins. Similar matrices were also constructed containing immobilized GST-p85~-N-terminal SH2 domain (GST-p85ct- N-SH2), GST-p85a-Bcr homology region (GST-p85a- Bcr), unfused GST, or glutathione-Sepharose alone. Figure 1A shows that several proteins bound specifically to the GST fusion proteins containing different domains from p85a (Figure 1A, lanes 5-10) but not to GST/glutathione-Sepharose or glutathione-Sepharose alone (Figure 1A, lanes 1-4). Notably, a 100 kd protein (pl00) was seen to bind only to the affinity matrix containing the GST- p85~-SH3 fusion protein (Figure 1A, lanes 9 and 10). Fur- ther experiments indicated that pl00 was abundant in bovine brain and bound very tightly to this affinity column. Several other GST-SH3 domain fusion proteins were prepared (GST-P67-C-terminal SH3 [GST-P67-C-SH3], GST-P67-N-terminal SH3, GST-P47-C-terminal SH3, GST-neuronal src-SH3 [GST-n-src-SH3], GST-cellular src-SH3 [GST-c-src-SH3], GST-PLCy-SH3 and GST- rasGAP-SH3) and used as affinity matrices to screen bovine brain extracts for additional associated molecules. We found that GST-SH3 domains, but not GST alone, specifically associated with a number of proteins (Figure 1B). Some of these proteins (p55, p70, pl00, and p150) were purified in quantities sufficient for protein sequencing by high resolution methods (Totty et al., 1992). An analysis of the pl00 SH3 domain-binding molecule is presented in this paper.

SH3 Domains Bind the GTPase Dynamin 27

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Figure 1. Proteins from Bovine Brain Extracts Bound to Recombinant GST Domains of p85a and SH3 Domains from Various Proteins (A) Proteins bound to SH2, SH3, and Bcr homology domains of p85a. Either 0.02% (v/v) (lanes 1, 3, 5, 7, and 9) or 1.0% (v/v) (lanes 2, 4, 6, 8, and 10) Triton X-100 extracts of bovine brain were incubated with glutathione-Sepharose beads (lanes 1 and 2) or glutathione- Sepharose beads coupled to GST (lanes 3 and 4), GST-p85a-Bcr (lanes 5 and 6), GST-p85a-N-SH2 (lanes 7 and 8), or GST-p85a-SH3 (lanes 9 and 10). The beads were washed extensively, and associated proteins were eluted by boiling in Laemmli sample buffer. Released proteins were resolved by SDS--PAGE on a 12.5% gel and visualized by silver staining. Arrows indicate band~ corresponding to GST and GST fusion proteins. (B) Proteins bound to different GST-SH3 domains. GST or GST-SH3 fusion proteins were coupled to glutathione-Sepharose beads and incubated with a 1% Triton X-100 extract of bovine brain. After extensive washing, bound proteins were eluted from the beads by boiling in Laemmli sample buffer. Released proteins were resolved by SDS- PAGE on a 10% gel and visualized by silver staining. Lanes: 1, GST-P67-C-SH3; 2, GST-P67-N-SH3; 3, GST-P47-C-SH3; 4, GST- n-src~SH3; 5, GST-c-src-SH3; 6, GST-PLCy-SH3; 7, GST-rasGAP- SH3; 8, GST-p85a-SH3; and 9, GST alone.

1 MGNRGMED L I P LVNRLQDAF SAI GQNADLD LP Q I AWGGQ SAGK S SVLEN 50

51 FVGRDF LP RG SG I VTRRP L%rLQLVNS T T EYAEFLHCKGKKF TDFEEVRLE i00 (Pepl) GXSP%*PI~ (Pep2) % ~ P

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151 D I EFQ I RDMLMQFVTKENCL I LAVSPAN SD LANS DItLK IAKEVDPQGQRT 200 (Pep3) GXIG~

201 IGVI TKLDLMDEGTDARDVLENKLLPLRRGYI GVVNRSQKD IDGKKD ITA 250 (Pep4) FFLSRPSYR (Pep5) L

251 ALAAERKFF LSHP S YRHLADRMGTP yLQKVLNQQLTNH I RDTLPGLRNKL 300 QSQLLSZEK

301 Q SQLL S I EKEVDEYKNFRPDDPARKTKALLQMVQQFAVDFEKR I EG SGDQ 350

351 IDTYELSGGARINRIFHERFPFELVK~FDEKELRREI SYAI KN IHGIRT 400

401 GLF TP DLAFEATVKKQVQKLKEP S I KCVDMVVSELT S T I RKC SEKLQQYP 450

451 RLREEMER IVT T H I REREGRTKEQVMLL I D I ELAY~TNHEDF I GFANAQ 500

501 QRSNQMNKKKTSGNQDE ILVIRKGWLTINNIGIMKGGSKEYWFVLTAENL 550 (Pep6) ~SVDNLK (Pep7) HXFAIJ~

551 SWYKDDEEKEKKYMLSVDNLKLRDVEKGFMS S KH I FALFNTEQRNVYKD Y 600

601 RQLELACETQEEVD SWKASF LRAGVYP ERVGDKEKAS ETEENGS D SFMH $ 650

651 M~gP QLERQVET I RNLVD S YMAIVNKTVRD LMP KT I MHLMINNTKE F I F SE 700

701 LLANLYSCGDQNTLMEESAEQAQRRDEMLRMyHALKEALS I I GD INTTTV 750

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751 S TPMP P PVDD SWLQVQSVPAGRRSP T S SP TP QRRAPAVP PA/~PGS RGPAP 800 N 2 - -

801 GPPPAGSALGGAPpVPSRPGASPDPFGPPPQVPSRPNRAPPGVPRITISDP 851 N3- N 4 - -

Figure 2. Alignment of pl00 Tryptic Peptides with the Rat Dynamin Protein Sequence Peptide sequences obtained from tryptic digests of pl00 are indicated in bold above the appropriate sequence of dynamin. Potential proline- rich SH3 domain-binding sites that fall into two clusters of overlapping sequences are underlined and identified as N1-N5. Peptides derived from dynamin and used in this study are overlined and identified as P1 and P2.

Ident i f icat ion o f p l 0 0 As D y n a m i n To characterize further the p l00 SH3 domain-binding protein, a larger scale affinity purification was carried out using the GST-p85a-SH3 domain fusion protein, as described in Experimental Procedures. Affinity purified p100 was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto a polyvinylidene difluoride membrane, and then subjected to N-terminal protein sequence determination. This showed the N-terminus of p l00 to be blocked. Affinity purified p l00 was therefore digested in the gel with trypsin after SDS-PAGE and staining with Coomassie blue. Tryptic peptides were resolved by high pressure liquid chromatography and subjected to protein sequence determination. Seven unambiguous sequences (Pepl-Pep7) were obtained (Figure 2). These peptides were used to search the protein sequence data bases using the FASTA program (UWGCG Package; Dev- ereux et al., 1984). As shown in Figure 2, all seven peptides were identical to the equivalent sequence of rat dynamin (Obar et al., 1990), thus identifying the p l00 SH3 domain- binding protein as bovine dynamin.

Binding of Dynamln to a Panel of R e c o m b i n a n t SH3 D o m a i n s The data presented in Figure 1B show that the p l00 protein in the crude brain extracts was able to bind to a number of different GST-SH3 domain fusion proteins. To investi- gate this observation further, the SH3 domains from PLCT, c-src, n-src, fyn, fgr, rasGAP, crk, spectrin, c-src kinase

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Figure 3. Binding of Dynamin to Different GST-SH3 Domain Fusion Proteins GST-SH3 fusion proteins were coupled to glutathione-Sepharose beads end incubated with partially purified rat brain dynamin. The beads were washed extensively and bound proteins eluted by boiling in Laemmli sample buffer. Released proteins were resolved by SDS- PAGE on a 10% gel and visualized either by staining with Coomassie blue (A) or by immunoblotting with anti-dynamin antiserum (B). Lanes: 1, GST alone; 2, GST-p85a-SH3; 3, GST-rasGAP-SH3; 4, GST- PLCT-SH3; 5, GST-c-src-SH3; 6, GST-n-src-SH3; 7, GST-P47-C- SH3; 8, GST-P67-N-SH3; 9, GST-P67-C-SH3; 10, GST--crk-SH3; 11, GST-fgr-SH3; 12, GST-spectrin-SH3; 13, GST-Grb2-N-SH3; 14, GST-Grb2-C-SH3; 15, GST-Grb2 (whole protein); 16, GST-fyn- SH3; and 17, GST-csk-SH3.

(csk), the 47 kd (C-terminal SH3 domain) and 67 kd (N- and C-terminal SH3 domains) subunits of the NADPH oxidase complex, and the N- and C-terfninal SH3 domains from Grb2 were all expressed as GST fusion proteins. The en- tire Grb2 protein, expressed as a GST fusion protein, was also used in these studies. Figure 3 shows that dynamin exhibited strong binding to the intact GST-Grb2 fusion protein and to the SH3 domains derived from PLCT, Grb2 (N-terminal), and p85a. Binding was assessed only qualitatively: as strong (e.g., PLCT), weak (e.g., C-terminal domain of P67), or not detectable (e.g., rasGAP). Dynamin exhibited an intermediate degree of binding to the SH3 domains of pp60 ~'r~, fyn, fgr, and to the C-terminal Grb2 SH3 domain. Only trace amounts of dynamin were found

to be associated with the C-terminal SH3 domain of P67 NADPH oxidase subunit, and there was no detectable binding to the GST-SH3 domain fusion proteins derived from rasGAP, n-src, P47 (C-terminal), P67 (N-terminal), crk, spectrin, or csk, or to GST alone. To exclude the possibility that lack of binding of some of these domains was due to incorrect folding, the GST-n-src-SH3 domain, which does not bind dynamin, was subjected to one- dimensional nuclear magnetic resonance analysis. This demonstrated that this domain was folded (data not shown). These results indicated that dynamin exhibits preferential binding to a subset of recombinant SH3 domains. Individual SH3 domains from proteins possessing two SH3 domains (e.g., P67 and Grb2) also showed differ- ential binding specificities for dynamin, at least when expressed as GST fusion proteins.

It has been reported that GST fusion proteins exist as dimers and that this might affect the observed interactions with potential SH3 domain-binding proteins (Panayotou et al., 1993). To address this question, we adopted two approaches. First, GSTIGST-p85a-SH3 heterodimers were prepared by a process of denaturation and renatu- ration as described by Panayotou at al. (1993). These were then compared with the original GST-p85a-SH3/GST- p85a-SH3 homodimers for their ability to associate with dynamin. No difference in binding was observed (data not shown). Secondly, a monomeric maltose-binding protein- p85a-SH3 domain fusion protein was prepared, and its binding characteristics were compared with those of the dimeric GST-p85a-SH3 fusion protein. Again, both proteins were found to bind to dynamin with similar efficiencies, suggesting that the fusion partner has little or no effect on the binding characteristics of the recombinant SH3 domains used in this study (data not shown).

Analysis of the SH3 Domain-Binding Site in Oynamin Like dynamin, the 3BP1 protein has been shown to bind to different recombinant GST-SH3 domains with varying affinities (Cicchetti et al., 1992). A putative SH3 domain- binding site was identified in 3BP1 and was localized to a stretch of 10 amino acids (Cicchetti et al., 1992). Follow- ing a mutagenesis study, the motif XPXXPPPZXP was suggested as the basis for Abl SH3 domain-binding to 3BP1 (Ren et al., 1993). A putative binding site in mSosl has also been predicted (VPVPPPVPPRR; Bowtell et al., 1992; J. Downward, personal communication). Analysis of the dynamin sequence revealed the presence of up to five putative SH3 domain-binding sites related to these sequences, located in two clusters at the C-terminus of the molecule (see Figure 2). A 21 residue synthetic peptide containing the N 1 site sequence (peptide P 1, PAVPPARP- GSRGPAPGPPPAG; see Figure 2) had been synthesized earlier for structural studies and was shown to bind to the recombinant p85a-SH3 domain with a dissociation constant of the order of 3 x 10-4 M (Booker et al., 1993). The P1 peptide was able to inhibit dynamin binding to the GST-SH3 domains derived from PLC7 and p85a when used at micromolar concentrations (Figure 4A). The P1 peptide was unsuitable for preparing an affinity column,


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Figure 4. Analysis of the SH3 Domain-Binding Site in Dynamin (A) Inhibition of dynamin binding to GST-p85a-SH3 and GST-PLCy- SH3 domain fusion proteins by the dynamin-derived peptide P 1 (PAVP- PARPGSRGPAPGPPPAG). GST-p85a-SH3 (lanes 1-5) or GST- PLCy-SH3 (lanes 7-11) coupled to glutathione-Sepharoso beads were incubated with increasing amounts of peptide P1 before partially purified dynamin was added to the reaction mixture, lanes: 1 and 7, no peptide; 2 and 8, 5 p.M peptide; 3 and 9, 25 p.M peptide; 4 and 10, 125 p.M peptide; 5 and 11, 125 I~M control peptide (poly-L-proline; Sigma), 6, protein standards. (B) Binding of different GST-SH3 domains to the dynamin-derived peptide P2. Peptide P2 (SPTPQRRAPAVPPARPGS) coupled to Acti- gel resin was incubated with purified GST-SH3 domain fusion proteins. The beads were washed extensively and bound proteins eluted by boiling in Laemmli sample buffer. Released proteins were resolved by SDS-PAGE on a 10% gel and visualized by staining with Coomas- sie blue. Lanes: 1, GST alone; 2, GST-p85a-SH3; 3, GST-rasGAP- SH3; 4, GST-PLCy-SH3; 5, GST-O-src-SH3; 6, GST-n-src-SH3; 7, GST-P47--C-SH3; 8, GST-P67-N-SH3; 9, GST-P67-C-SH3; 10, GST--crk-SH3; 11, GST-spectrin-SH3; 12, GST-fgr-SH3; 13, GST- Grb2-N-SH3; 14, GST-Grb2-C-SH3; 15, GST-Grb2 (whole protein); 16, GST-fyn-SH3; and 17, GST--csk-SH3.

owing to the presence of an N-terminal proline residue. Therefore, to characterize this potential SH3 domain- binding sequence further, a second peptide containing the N1 site, P2 (SPTPQRRAPAVPPARPGS), was synthe-

sized and coupled to an Actigel matrix, and its ability to bind various SH3 domains and SH3 domain-containing proteins was determined. As shown in Figure 4B, there is a correlation between the observed specificity of binding of the different GST-SH3 domain fusion proteins to the P2 peptide column and their specificity of binding to the intact dynamin protein.

Analysis of the SH3 Domain-Binding Sites in 3BP1 and mSosl Short peptides, corresponding to the Abl SH3 domain- binding site in 3BP1 (RAPTMPPPLPPVPPQPAR; Cic- chetti et al., 1992; Ren et al., 1993) and to a putative binding site in mSosl (SKGTDEVPVPPPVPPRR; Bowtell et al., 1992; J. Downward, personal communication) were synthesized and coupled to an Actigel matrix so we could examine their binding specificities for the panel of SH3 domains. Both of these peptides revealed binding to a distinct subset of the panel of SH3 domains (Figures 5A and 5B). The SH3 domain from pp60 ~'sr¢ and the whole Grb2 protein (which possesses two SH3 domains) were shown to exhibit strong binding to the 3BP1 peptide in comparison with the SH3 domains from p85a, PLCy, fgr, and fyn. However, neither the N-nor C-terminal SH3 domains from Grb2 individually exhibited a significant interaction with the immobilized 3BP1 peptide. This observation may suggest that the presence of two or more SH3 domains within a protein might increase its binding affinity for the associated molecules. The peptide from mSosl bound intact Grb2 protein, the recombinant GST-SH3 domains from p85a, PLCy, crk, and fyn, and the N- and C-terminal SH3 domains of the Grb2 protein with similar efficiencies. Weak binding of the pp60 ~Src and the fgr SH3 domains, as well as very weak binding of neuronal src SH3 domain, was also observed with this peptide. The binding of SH3 domains to all of these peptides is summarized in Table 1.

Purification and Characterization of Rat Brain Oynamin Biochemical characterization of dynamin had suggested that it binds tightly to microtubules and possesses an intrinsic microtubule-stimulated GTPase activity (Shpetner and Valise, 1992; Maeda et al., 1992). A microtubule- binding site has been localized to the C-terminal region of dynamin (Valise, 1992), which contains the proline-rich sequences thought to be involved in SH3 domain binding. To assess the influence of SH3 domain binding on dynamin GTPase activity, rat brain dynamin was purified to near homogeneity using a modification of published purification procedures.

Two purification methods for dynamin have been described, based either on association with microtubules in the presence of GTP and AMP-PNP (Shpetner and Vallee, 1989) or on affinity purification using ATP-agarose (Scaife and Margolis, 1992). A 1 day purification method was de- veloped from these that allowed the purification of large amounts of rat brain dynamin. A diethylaminoethyl (DE-52) cellulose column effectively separated dynamin from en- dogenous tubulin and microtubule-associated proteins

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Figure 5. Binding of Different GST~SH3 Domains to Proline-Rich Pep- tides Derived from 3BP1 and mSosl Synthetic peptides from 3BP1 (RAPTMPPPLPPVPPQPAR) and mSosl (SKGTDEVPVPPPVPPRR) were coupled to Actigel and incubated with purified GST-SH3 domain fusion proteins. The beads were washed extensively and bound proteins eluted by boiling in Laemmli sample buffer. Released proteins were resolved by SDS-PAGE on a 10% gel and visualized by staining with Coomassie blue. The proteins associated with the immobilized 3BPl-derived peptide are shown in (A) and those bound to the mSosl-derived peptide in (B). Lanes: 1, GST alone; 2, GST-p85a-SH3; 3, GST-rasGAP-SH3; 4, GST-PLCy- SH3; 5. GST-c-src-SH3; 6, GST-n-src-SH3; 7, GST-P47-C-SH3; 8, GST-P67-N-SH3; 9, GST-P67-C-SH3; 10, GST-crk-SH3; 11, GST- spectrin-SH3; 12, GST-fgr-SH3; 13, GST-Grb2-N-SH3; 14, GST- Grb2-C-SH3; 15, GST-Grb2 (whole protein); 16, GST-fyn-SH3; and 17, GST-csk-SH3. •

(Figure 6, lane 3). The Mono S column achieved efficient separation of dynamin from the bulk of the remaining proteins (Figure 6, lane 4). Immunoreactive dynamin was eluted from the Mono S column at 0.2-0.25 M NaCI. A third column of ATP-agarose yielded preparations of essentially homogeneous dynamin. Each step of purification was monitored by immunoblotting with specific anti- dynamin antibodies (data not shown). By this protocol,

Figure 6. Purification of Rat Brain Dynamin Dynamin was purified from 10 rat brains as described in Experimental Procedures. Purification was monitored by SDS-PAGE, and proteins were visualized by staining with Coomassie blue. The 100 kd bovine dynamin is indicated (D100). Lanes: 1, protein standards (180, 116, 84, 58, 48, 36, and 26 kd; Sigma); 2, 50K supernatant of rat brain extract; 3, DE-52 flow-through; 4, pool of peak fractions following Mono S anion exchange chromatography; 5, ATP-agarose affinity chromatography.

approximately 350 ~g of dynamin was purified from 10 rat brains. When added to immobilized GST-SH3 fusion proteins, purified dynamin bound the same spectrum of SH3 domains as the dynamin present in crude rat or bovine brain extracts.

Stimulation of Dynamin GTPase Activi ty by S H 3 Domains As can be seen in Figure 7, a number of the panel of GST- SH3 domain fusions used in our binding studies were able to stimulate the GTPase activity of dynamin to varying degrees. GST-SH3 domain fusion proteins and GST- Grb2 were assayed without dynamin and found not to be contaminated with bacterial GTPases. There was no effect of GST-SH3 fusion proteins on nucleotide binding to dynamin (data not shown), which suggests that these domains do not act as exchange factors to stimulate GTP hydrolysis on dynamin. Purified rat brain dynamin had no detectable intrinsic GTPase activity when assayed alone under conditions described but was strongly stimulated by the addition of taxol-polymerized microtubules (Figure


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=

a b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 7. Stimulation of Dynamin GTPase Activity by SH3 Domains Purified dynamin (0.5 p.g) was incubated with or without GST-SH3 domain fusion proteins (2 p.g) in the presence of [a-=P]GTP for 1 hr at 37°C. The extent of GTP hydrolysis after I hr was assessed by thin layer chromatography. If the formation of GDP was less than 5% of the amount of [a-32P]GTP present in the initial reaction mixture, it was regarded as insignificant. Lan~s: a, dynamin alone; b, dynamin plus GST-SH3 domain storage buffer; 1-17, dynamin plus GST-SH3 domain fusion proteins (1, GST alone; 2, GST-p85a-SH3; 3, GST-ras- GAP-SH3; 4, GST-PLCy-SH3; 5, GST--c-src-SH3; 6, GST-n-src- SH3; 7, GST-P47-C-SH3; 8, GST-P67-N-SH3; 9, GST-P67-C-SH3; 10, GST-crk-SH3; 11, GST-fgr-SH3; 12, GST-spectrin-SH3; 13, GST-Grb2-N-SH3; 14, GST-Grb2-C-SH3; 15, GST-Grb2 [whole protein]; 16, GST-fyn-SH3; and 17, GST-csk-SH3).

8A)• The initial rate of GTP hydrolysis induced by taxol- polymerized microtubules was 980 ± 25 nmol/min/mg, which agrees well with previously published values (Shpetner and Vallee, 1992). When added to purified rat brain dynamin, the GST-Grb2 (whole protein) fusion protein and the GST-SH3 domain fusion proteins derived from pp60 ~'~, fgr, and fyn were found to stimulate mark- edly the intrinsic dynamin GTPase activity (Figure 8B)• Initial rates of hydrolysis induced by GST-Grb2 protein or by the GST-c-src-SH3, GST-fgr-SH3, and GST-fyn- SH3 domains were 350 ± 15, 195 ± 10, 180 ± 5, and 95 ± 10 nmol/min/mg, respectively. Taxol-polymerized microtubule stimulation of dynamin GTPase activity was observed to be greater than activation by GST-SH3 domains or GST-Grb2• A much lower level of activation was observed in the presence of the p85ct, PLCy, and the C-terminal Grb2 SH3 domain fusion proteins, although all of these domains bound to dynamin efficiently• GST-SH3 domains that did not bind dynamin failed to stimulate its GTPase activity significantly.

Discussion

An affinity chromatography approach was used to identify several SH3 domain-binding proteins from bovine brain extracts. Purification and microsequencing of one of these proteins, pl00, showed it to be identical to the previously characterized protein dynamin (Obar et al., 1990). Dy- namin was originally purified from bovine brain as a micro-

~ 100

!. 26 - -

J m f n

• Dynimln plum mk:cotubulee

• I)ynlmln 14ene

Mlorotul~Jkm alone

7s

H

0

m l n

Figure8. Time Course of GTP Hydrolysis Induced by Taxol- Polymerized Microtubules or Different GST-SH3 Domain Fusion Pro- teins Two micrograms of GST-SH3 domain fusion proteins (B) or taxol- polymerized microtubulas (A) were incubated with purified dynamin (0.5 p.g) in the presence of [a-a=P]GTP for 60 rain at 37°C. Aliquots were taken at 0, 1, 5, 15, 30, and 60 min, and the reaction products were resolved by thin layer chromatography. Radioactive spots corresponding to GDP were quantitated using a phosphorimager (Molecular Dynamics).

• Ccrdml

I G$T • OSTt88o~11H3

GST/GAP-SH3 o~rlPLC~SFI3

- - - - . ~ - - GST,'GR B2

i Ml©rolububal

tubule-associated molecule, and early biochemical analysis suggested that it might act as a motor protein for microtubules (Shpetner and Vallee, 1989). Cloning and characterization of the dynamin cDNA revealed the presence of a consensus guanine nucleotide-binding site (Obar et al., 1990). Recently, dynamin has been shown to possess microtubule-stimulated GTPase activity, and a microtubule-binding site has been localized to a C-terminal 10 kd proteolytic fragment of the protein (Vallee, 1992)• Northern blot analysis demonstrated human dynamin to be expressed in all tissues studied, with exceptionally high levels in the brain (van der Bliek et al., 1993). Dynamin shows extensive similarity throughout its length to the product of the Drosophila gene shibire (van der Bliek and Meyerowitz, 1991). It also shows sequence similarity through its GTP-binding domain to the mammalian Mxl and Mx2 proteins, which promote interferon-inducible re- sistance to viral infection (Rothman et al•, 1990), and to a Saccharomyces cerevisiae protein that has been inde- pendently isolated as vacuole protein-sorting protein 1 and as SP015, which is implicated in spindle pole separation during meiosis (Yeh et al., 1991; Vater et al., 1992). To- gether, these proteins form a new group of related, large GTP-binding proteins. Their precise function, however, remains unclear.

Dynamin was found to bind specifically to a subset of GST-SH3 domain fusion proteins. Binding was only as-

Cell 32

Table 1. Summary of the Binding Data of Recombinant GST-SH3 Domain Fusion Proteins to Different Protein-Rich Synthetic Peptides and Whole Dynamin

Dynamin Fusion Protein P2 3BP1 Sos Dynamin GTPase

GST . . . . . GST-85a ++ + ++ ++ + G S T - G A P . . . . . GST-PLC7 ++ + +++ +++ +/- GST-src ++ ++ + + +++ GST-n-src - - +/- - - GST-P47-C-SH3 . . . . . GST-P67-N-SH3 . . . . . GST-P67-C-SH3 + - - +/- - GST-crk +/- - ++ - - GST-spectrin +/ . . . . . GST-fgr + + ++ + ++ GST-GRB2-N-SH3 +++ +/- +++ +++ - GST-GRB2-C-SH3 ++ +/- +++ ++ +I- GST-GRB2 full length +++ ++ +++ +++ +++ GST-fyn + + ++ + + GST-csk . . . . .

Shown are the GST-SH3 domain fusion proteins that bind to the dynamin-derived peptide P2 (column 1), 3BP1 peptide (column 2), Sos peptide (column 3), and whole dynamin (column 4). Data can be ex- trapolated from Figures 3, 4B, 5A, and 5B. Strong binding is indicated by three plus signs, an intermediate degree of binding by two, weak binding by one, very weak binding by one minus and one plus sign, and no detectable binding by one minus sign. Column 5 summarizes the data from Figures 7 and 8 on activation of the dynamin intrinsic GTPase activity by the different recombinant SH3 domains.

sessed qualitatively but could be categorized as strong (e.g., GST-PLCy-SH3), intermediate (e.g., GST-fgr - SH3), weak (e.g., GST-P67-C-SH3), or not detectable (e.g., GST-rasGAP-SH3). This data is summarized in Ta- ble 1. Preliminary experiments examining real time interactions between purified rat brain dynamin and the GST- SH3 domain fusion proteins of p85~ and pp60 ~s~ using the BIAcore biosensor indicated affinities in the nanomolar range for this interaction (G. Panayotou, unpublished data).

In this study, we have shown the binding of SH3 domains to two distinct sets of proline-rich sequences, the proto- types of which are the mSosl and 3BP1 peptides (Table 2). The 3BP1- and mSosl-derived peptides exhibited selective binding toward a distinct, but overlapping, spectrum of recombinant SH3 domains (compare with Table 1). Renet al. (1993) have described a mutagenesis analysis of the Abl SH3 domain-binding site of the 3BP1 protein demonstrating the requirement of certain amino acid residues for specific association. This ~.pproach allowed them to define a consensus sequence required for SH3 domain binding to the proline-rich motif as XPXXPPPZXP. Proline residues at positions 2, 7, and 10 in this motif were found to be essential for binding. Amino acid residues at positions 1 and 9 were also found to affect binding when mutated but were not highly conserved among the SH3 domain- binding sites examined. The mSosl peptide used in this study contains proline residues with a spacing distinct from that found in the 3BPl-binding site, having a motif

Table 2. Alignment of Putative SH3 Domain-Binding Sites

Protein Amino Acids Sequence

Human/rat dynamin N1 (786-793) PAVPPARP mSOS1 (1151-1158) PVPPPVPP Consensus PXXPPXXP

Human/rat dynamin N2 (781-789) PQRRAPAVP Human/rat dynamin N4 (828-836) PPPQVPSRP Drosophila Shibire 1 (803-811) PAPA I PNRP Drosophila Shibire 2 (811-819) PGGGAPPLP

Human/rat dynamin N3 (825-833) PFGPPPQVP Human/rat dynamin N5 (836-844) PNRAPPGVP Murine 3BP1 (267-275) PTMPPPLPP Consensus PXXXPPXXP

Rat and human dynamin, the Drosophila homolog of dynamin, shibire, 3BP1, and mSosl have been divided into two groups of sequences containing related but distinct proline-rich motifs. Putative proline core consensus sequences are presented in bold underneath each group and probably indicate the structural requirements for SH3 domain binding rather than SH3 domain-specific recognition motifs, in the case of 3BP1, the amino acid position numbers count from the beginning of the partial cDNA sequence given by Cicchetti et al. (1992).

of the form XPXXXPXXP (Table 2). This indicates that SH3 domains may tolerate some flexibility in the spacing of the important proline residues in the binding motif. Amino acids located around the proline residues in both motifs appear not to be conserved, and this may indicate that they define the specificity required for interaction of this motif with distinct SH3 domains (Table 2). On the basis of sequence similarity to these two consensus motifs, five proline-rich peptide sequences that may be SH3 domain- binding sites within dynamin could be predicted (N1-N5; Table 2). These sequences occur in two clusters within the C-terminal 100 amino acids of dynamin (see Figure 2) and are conserved in both the human and rat proteins. The Drosophila homolog of dynamin, the shibire gene product, also possesses two proline-rich related sequences near its C-terminus (van der Bliek and Meyero- witz, 1991; Table 2). We synthesized two peptidas, P1 and P2, that contained the N1 (mSosl-related) binding site from dynamin. Peptide P2 showed the same binding specificity for the recombinant SH3 domains as the intact dynamin protein, while peptide P1 was able to inhibit dynamin from binding to the GST-SH3 domains from p85~ and PLCy. However, a high concentration of the dynamin- derived peptide P1 (125 mM) was necessary to inhibit binding. This may reflect a requirement for the binding site to exist in a particular conformation for high affinity binding, or alternatively may mean that dynamin contains a number of distinct functional SH3 domain-binding sites. Together, these data suggest that SH3 domain-binding motifs may be more varied in sequence than has been suggested to date. A more detailed study will be required to address this issue; it will be complicated in the cases of dynamin and 3BP1 because of the presence of multiple, overlapping potential binding sites (see Figure 2).

All of the proposed SH3 domain-binding sequences that have been shown to bind SH3 domains have one thing in


common: the presence of a diproline motif. This may be significant, as consecutive proline residues are known to restrict the conformation of the preceding residues, thereby inducing a left-handed helical twist in aqueous solution (Creighton, 1984). The importance of such a twist is that it allows the side chains of residues at positions i and i+3 (i.e., the residues on either side of the diproline motif) to adopt similar orientations on one side of the peptide chain. This is not possible with commonly observed forms of secondary structure such as a helix (i and i+4) or I~ sheet (i and i+2). An example of such a conformation can be found at positions Pro-8 and Pro-9 in the crystal structure of bovine pancreatic trypsin inhibitor (Bode et al., 1978). Given the high number of aromatic residues within the proposed binding surface of the p85a SH3 domain (Booker et al., 1993), it is interesting to observe the nature of the side chain packing within bovine pancreatic trypsin inhibitor. Pro-9 can be seen to stack against the aromatic ring of Phe-22 and makes additional perpendicu- lar contacts with the aromatic ring of Phe-33. It is possible, therefore, that the specificity of the SH3 domain-ligand interactions stem from the composition of the residues on either side of the diproline motif. This view is complicated by the occurrence, in a number of proposed SH3 domain- binding peptides, of more than one diproline motif or the presence of triproline sequences (Table 2). A combination of mutagenasis, binding kinetics, and structural determination studies may be required to define the ligand more fully.

Dynamin has been shown to possess an intrinsic GTPase activity that is stimulated by the binding of microtubules to its C-terminus (Shpetner and Vallee, 1992; Vallee, 1992). Since the putative SH3 domain-binding sequences also occur in this region, the effect of SH3 domain association on dynamin GTPase activity was investigated. A subset of the GST-SH3 domain fusion proteins that bind to dynamin were also able to stimulate its intrinsic GTPase activity significantly. The most potent activators of the GTPase activity were the GST-c-src-SH3 domain and GST-Grb2 (which has two SH3 domains). A lower level of activation was induced by SH3 domain fusion proteins derived from p85a, PLC7, and the C-terminal SH3 domain of Grb2. There was no clear correlation between the appar- ent binding affinity of a particular SH3 domain fusion protein for dynamin and its ability to stimulate the rate of GTP hydrolysis on dynamin (see Table 1). For example, the GST-fgr-SH3 domain fusion protein exhibits relatively weak binding to dynamin, yet is a potent activator of the dynamin GTPase activity, while the SH3 domain of PLC7 shows strong binding to dynarnin but does not enhance its GTPase activity. We cannot exclude the possibility that the nonquantitative binding assay we have used in this study does not reflect the precise affinities displayed by these molecules. Alternatively, different SH3 domains may bind to distinct proline-rich sites within the C-terminal region of dynamin, and the observed activation of the dynamin GTPase activity may require binding to one or more specific sites. We are currently investigating further the binding specificities of the multiple putative SH3 domain-

binding sites in dynamin, which are located at the C-terminus of the molecule, in an attempt to locate such a region more precisely.

The observation that SH3 domains are able to stimulate the rate of hydrolysis of GTP on dynamin directly is the first indication that not only are these domains involved in protein-protein interactions, but they can also regulate the function of associated molecules. A potential link between SH3 domains and regulators of small G proteins has been suggested before (Pawson and Gish, 1992; Mayer and Baltimore, 1993). The deduced amino acid sequence of 3BP1 contains a region showing sequence similarity to a Bcr homology domain found in n-chimerin, P190, and rhoGAP (reviewed by Fry, 1992). The bacterially expressed Bcr homology domains from n-chimerin, rhoGAP (Diekmann et al., 1991), and P190 (Settleman et al., 1992) stimulate the GTPasa activity of rac and rho, suggesting that 3BP1 may also function as a small G protein-specific GAP (Cicchetti et al., 1992). It is, therefore, interesting to speculate that the binding of SH3 domains to 3BP1 might also regulate its GAP activity. Thus, the regulation of GTPasa activity either directly or indirectly, via a GAP-like molecule, may be a general feature of SH3 domain function.

What still remains unclear, however, is the role, if any, that dynamin plays in receptor-stimulated signal transduction pathways mediated via SH3 domains. Studies to date on mammalian dynamin have not proved particularly help- ful in providing any rationale for SH3 domain-dynamin interactions (reviewed by Vallae, 1992). Some ideas about the cellular function of dynamin have come from studies on the Drosophila temperature-sensitive dynamin mutant shibire. When shifted to the nonpermissive temperature, the shibire defect causes paralysis by inhibiting neuro- transmitter endocytosis, most likely at the level of the pinching off from the plasma membrane of neurotransmit- tar-containing vesicles (Masur et al., 1990; Chen et al., 1991; van der Bliek and Meyerowitz, 1991). Recently, it has been demonstrated that point mutations in the GTP- binding domain of human dynamin specifically inhibit early events in receptor-mediated endocytosis (van der Bliek et al., 1993). The yeast homolog vacuole protein-sorting protein I has also been implicated in events involving vesicle transport (Vater et al., 1992), and mammalian dynamin was recently suggested to have a potential role in clathrin- coated vesicle function (Carter et al., 1993; van der Bliek et al., 1993; Schmid, 1993). It is difficult to see a direct correlation between neu rotransmitter endocytosis, vesicle protein sorting in yeast, and signaling through SH3 domains, although all three processes do involve cytoskeletal interactions, vesicle movements, and GTP hydrolysis. The next important steps are clearly to identify those SH3 domain-containing proteins that are able to interact with dynamin in vivo. In this regard, coimmunoprecipitation of both PLC~, and P85a with dynamin has recently been observed in PC12 cells (R. Scaife and B. Margolis, personal communication). Additionally, it will be important to determine into which of the post-receptor signaling pathways this protein fits.

Cell 34

Experimental Procedures

Ceil Lines and mRNA Isolation U937 cells were maintained in RPMI medium containing 10% fetal calf serum. Total RNA from rat brain or U937 cells was isolated by the method of Chirgwin et al. (1979), reverse transcribed, and used in the polymerase chain reaction (PCR) amplification of the SH3 domain sequences of n-src (rat brain) and of the P47 and P67 subunits of NADPH oxidase (U937).

Bacterial Expression of the SH2 and Bcr-Homology Domains of p85a, and 14 Different SH3 Domains as GST Fusion Proteins Sequences corresponding to the SH3 domain (amino acids 1-86), Bcr homology region (amino acids 133-322), and N-terminal SH2 domain (amino acids 314-431) of the bovine p85a subunit of PI 3-kinase (Otsu et al., 1991) were amplified by PCR and cloned into the pGEX-2T expression vector (Pharmacia). BamHI and EcoRI sites were included at the ends of the N-terminal and C-terminal oligonucleotides, respectively, to facilitate cloning. Stop codons were introduced at the end of each of the cloned sequences 5' to the EcoRI site.

The source of the various SH3 domains used in this study were as follows, pGEX-lyn-SH3 (amino acids 86-139; Courtneidge et al., 1993), pGEX-csk-SH3 (amino acids 6-77), and pGEX-GRB2 (amino acids 1-217) have been described previously. Sequences coding for the SH3 domains of human rasGAP (amino acids 275-345), human PLC7 (amino acids 792-851), chicken pp60 ~"c (amino acids 84-148), human Grb2 (N- and C-terminal SH3 domains; amino acids 1-58 and 159-217, respectively), human fgr (amino acids 80-137), chicken spectrin (amino acids 967-1025), and crk (amino acids 369-429) were amplified by PCR from the corresponding cDNAs. SH3 domain-coding sequences of n-src (amino acids 84-154), P47 subunit of NADPH oxi- dasa (C-terminal SH3 domain; amino acids 226-286), and P67 subunit of NADPH oxidase (N- and C-terminal SH3 domains; amino acids 235- 300 and 458-517, respectively) were amplified by reverse transcription PCR (RT-PCR) using mRNA isolated from rat brain (n-src) or U937 cells (P67 and P47), PCR primers were designed with BamHI and EcoRI restriction sites and in-frame stop codons as described above. PCR-amplified sequences for each SH3 domain were cloned into the pGEX-2T (Pharmacia) expression vector using BamHI and EcoRI. The cloned sequences were sequenced using the Sequenase system (US Biochemical). pGEX-2T constructs were transformed into Escherichia coil XLI-Blue (Stratagene), and expression of GST fusion proteins was carried out as described previously (Smith and Johnson, 1988),

Affinity Purification of SH3 Domain-Binding Proteins Affinity resins for the isolation of SH3 domain-binding proteins were prepared by immobilizing the GST-SH3 domain fusion proteins on glutathione-Sepharose beads (Pharmacia) as described (Smith and Johnson, 1988). Similar resins were prepared using GST alone, GST- p85a-N-SH2, or GST-p85a-Bcr homology region to control for the specificity of interaction. Affinity resins were incubated with either 0.02% or 1% Triton X-100 extracts of total bovine brain for 2 hr at 4°C. After extensive washing, proteins bound to the resins were released, by boiling in SDS sample buffer, separated by SDS-PAGE, and visualized by silver staining.

Purification and Sequencing of pl00 Glutathione-Sepharose beads preloaded with approximately 50 I~g of the GST-p85a-SH3 fusion protein were incubated with a 1% Triton X-100 extract of bovine brain for 2 hr st 4°C. After extensive washing in incubation buffer containing 0.5 M NaCI, proteins were released from the affinity resin by boiling in sample buffer and were fractionated by SDS-PAGE. The region of the gel containing the pl00 protein was excised, and a digestion was performed in the gel slice with trypsin (J, Hsuan, unpublished data). Peptidas were separated by reverse-phase high pressure liquid chromatography and sequenced using a modified Applied Biosystems 477A sequencer (Totty et al., 1992).

Binding of Dynamin to GST-SH3 Fusion Proteins Two micrograms of GST-SH3 domain fusion proteins or GST alone was bound to glutathione-Sepharosa beads in buffer A (10 mM Tris- HCI [pH 7.5], 1% Triton X-100, 150 mM NaCI, 5 mM EDTA, 50 mM

NaF, 1 mM phenylmathylsulfonyl fluoride, and 100 Kallikrein inhibitor units of aprotinin per ml). The beads were then washed with buffer A to remove uncoupled GST fusion proteins. The amounts of GST fusion proteins used were normalized by Western blotting with GST-specific antibodies. Preloaded beads were incubated with partially purified rat brain dynamin (Mono S fraction). After extensive washing, the beads were boiled in sample buffer, and the released proteins were analyzed by SDS-PAGE.

Binding of Different SH3 Domains to Synthetic Peptldes from Dynamln, 3BP1, and mSosl Synthetic poptidas corresponding to putative SH3 domain-binding sequences in dynamin (P1, PAVPPARPGSRGPAPGPPPAG; P2, SPTPQRRAPAVPPARPGS) 3BP1 (RAPTMPPPLPPVPPQPAR), and mSosl (SKGTDEVPVPPPVPPRR) were synthesized on an Applied Biosystems 430A instrument using FMOC chemistry. Peptides were purified by reverse-phase high pressure liquid chromatography and coupled to Actigel resin (Sterogene Biosaparations, Inc.) according to the manufacturer's instructions. The peptide affinity resins were incubated in buffer A with 2 ~g of either purified GST-SH3 fusion proteins or GST. After extensive washing, bound proteins were eluted from the beads by boiling in sample buffer and analyzed by SDS-PAGE.

Inhibition of GST-SH3 Domaln-Dynamln Binding by the P1 Peptlde Two micrograms of either GST-p85a-SH3 or GST-PLCy-SH3 were coupled to glutathione-Sepharose beads in buffer A for 1 hr at 4°C. After washing to remove unbound proteins, the dynamin-derived peptide P1 was added to the beads at a final concentration of 5 ~.M, 25 ~M, or 125 I~M, Poly-L-proline peptide (Sigma Chemical Co.) was used at 125 I~M in the control reaction. After a 1 hr incubation, an equal amount of partially purified rat brain dynamin (Mono S fraction) was added to each reaction mixture. After a further 2 hr incubation, the beads were washed extensively with buffer A and boiled in SDS- PAGE sample buffer, and the amount of bound dynamin was assessed following SDS-PAGE.

Purification of Dynamln from Rat Brain Ten rat brains were homogenized in 40 ml of ice-cold buffer B (10 mM sodium phosphate [pH 6.9], 70 mM sodium glutamate, 2 mM EDTA, 2 mM MgSO4,1 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 1 p.g/ml pepstatin A, 10 i~g/ml leupepfin, and 100 Ulml aprotinin). All subsequent procedures were carried out at 4°C. The homogenate was centrifuged for 10 min at 10,000 rpm (Sorvall SS34 rotor) and the supernatant recentrifuged for 45 rain at 50,000 rpm in a Beckman TL-100 centrifuge (TLA 100.2 rotor). The supernatant was applied to a 30 ml DE-52 column (Whatman BioSystems, Inc.) preaquilibrated in buffer B. Flow-through material was loaded onto a 5 ml Mono S column equilibrated in buffer B. After extensive washing, bound proteins were eluted with a 0-500 mM NaCI gradient in buffer B. Peak fractions were combined and diluted with buffer B to give a final NaCI concentration of 50 mM. This material was loaded onto a 1 ml ATP- agarose column (Sigma) equilibrated in buffer B containing 50 mM NaCI. After washing, dynamin was eluted from the column with 0.5 M NaCI. Peak fractions were dialyzed against buffer B containing 50% glycerol and stored at-70°C. Each step of the purification protocol was monitored by immunobloting with anti-dynamin antisera. The antibody used (Ab 748) was raised against a bacterially expressed fragment of the Drosophila homolog of dynamin, shibirs (amino acids 350-610), which are highly conserved with rat dynamin (van der Bliek et al., 1993).

Purification of Tubulin and Preparation of Mlcrotubules Tubulin was purified from 5 rat brains as described previously (Williams and Lee, 1982). Taxol-polymerized microtubulas were prepared by incubation of tubulin with an equimolar amount of taxol (Sigma) at 30°C for 15 rain. Polymerized microtubulas were pe,eted by centrifu- gation at 55,000 rpm (TL-100 centrifuge, TLA 100.2 rotor) at 2°C for 30 rain.

Assay of Dynamln OTPase Activity Dynamin GTPase activity was assayed in a 50 I~1 reaction mixture


containing 1 Ilg of dynamin in 50 mM Tris-HCI (pH 8.0), 5 mM MgCI~, 0.1 mM dithiothreitol, 130 IIM GTP, and 13 nM [a-3=P]GTP (3 p.Ci, 3000 Cilmmol; Amersham Corp.) for 1 hr at 37°C. Taxol-polymerized microtubules (0.2 mg/ml) or GST-SH3 domain fusion proteins (0.1 mg/ml) were added to the reaction mixture to assess the effect on dynamin GTPase activity. Reactions were performed for the times stated in the appropriate figures and were terminated by heating sam- ples to 65°C for 10 rain. The products of the reaction were resolved by thin layer chromatography on polyethyleneimine-cellulose plates in 1.6 M LiCI. The extent of GTP hydrolysis was assessed by quantitat- ing GDP formation with a phosphorimager (Molecular Dynamics). Data expressed are the amount of [~P]GDP formed as a percentage of the amount of [ct-~P]GTP added initially to the reaction mixture.

Acknowledgments

We would like to thank Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA) for anti-dynamin antibodies, Drs. Sara Courtneidge and Manfred Koegl (EMBO, Heidelberg) for pGEX-fyn-SH3 and pGEX- csk-SH3, and Dr. Matti Saraste for pT7-spectrin-SH3 plasmids. We are also grateful to Dr. Peter Parker (ICRF, London) for PLC7 cDNA, Drs. Julian Downward and Sean Egan (ICRF, London) for rasGAP cDNA and pGEX-GRB2, Dr. Paul Brickell (University College, London) for fgr cDNA, Drs. Christine Ellis (Chester Beatty Laboratories, London) and Deborah Anderson (Saskatoon Cancer Center, Canada) for crk cDNA, and Dr. Kurt BollmeroHofer (Friedrich Miescher Institut, Basel) for pp60 ~'¢ cDNA. We give thanks to Drs. Sandra Schmid, Robert Margolis, Robin Scaife, Howard Shpetner, and Richard Vallee for valu- able discussion on dynamin and Drs. Peter Parker and Julian Down- ward for useful discussions and critical reading of the manuscript. Finally, we would like to thank Geoff Scrace for help with sequence analysis, Alistair Sterling for oligonucleotide synthesis, and Ram Ku- mar for technical assistance. I. G. thanks Imperial Chemical Industries and Amgen and G. W. B. thanks Imperial Chemical Industries for financial support.

Received May 6, 1993; revised July 14, 1993.

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The GTPase dynamin binds to and is activated by a subset of SH3 domains

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