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Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW - J. Cell Biol. (2000)

Bottom Line: Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia.Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle.These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

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The SH3 domain of cortactin binds dynamin. a, A cytosolic extract of NIH/3T3 cells was incubated with GST or GST–cortactin SH3 domain that was immobilized on glutathione Sepharose beads (lanes 1 and 2) or immunoprecipitated with the Dyn 2-specific antibody (lane 3). The bound proteins were fractionated and analyzed by blotting with an antidynamin mAb. As shown by the prominent dynamin band in lane 2, a substantial amount of dynamin was retained on the GST–cortactin SH3 domain beads as compared with the GST beads alone (lane 1). b, The GST pull-down assay was carried out in the absence or presence of one of the five synthetic peptides (P1–P5) at 0.1- or 1-mM concentrations. Binding of dynamin to the immobilized GST–cortactin SH3 domain was measured by blotting with an antidynamin mAb. Note that the P2 and P1 peptides were the most effective in blocking dynamin binding to the GST–SH3 domain beads (c). As a control the GST pull-down assay was carried out using a GST fusion protein containing the SH3 domain of PLCγ-1, in the absence (lane 1) or presence (lanes 2–6) of 1 mM of each of the five peptides. Binding of dynamin by the PLCγ-1 SH3 domain was assayed by blotting with antidynamin antibodies. As for the cortactin–SH3 domain-associated beads, peptides P2 and P1 inhibited dynamin binding, and some inhibition was demonstrated by the P4 peptide. d, Peptide sequence of the Dyn 2 COOH terminus. The four proline rich sequences are underlined. The bottom shows the sequences of the five synthetic peptides used to test the inhibition of SH3–dynamin interactions. The consensus sequence PXXPSRP was found to inhibit Dyn 2 binding to the GST–cortactin SH3 domain.
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Figure 2: The SH3 domain of cortactin binds dynamin. a, A cytosolic extract of NIH/3T3 cells was incubated with GST or GST–cortactin SH3 domain that was immobilized on glutathione Sepharose beads (lanes 1 and 2) or immunoprecipitated with the Dyn 2-specific antibody (lane 3). The bound proteins were fractionated and analyzed by blotting with an antidynamin mAb. As shown by the prominent dynamin band in lane 2, a substantial amount of dynamin was retained on the GST–cortactin SH3 domain beads as compared with the GST beads alone (lane 1). b, The GST pull-down assay was carried out in the absence or presence of one of the five synthetic peptides (P1–P5) at 0.1- or 1-mM concentrations. Binding of dynamin to the immobilized GST–cortactin SH3 domain was measured by blotting with an antidynamin mAb. Note that the P2 and P1 peptides were the most effective in blocking dynamin binding to the GST–SH3 domain beads (c). As a control the GST pull-down assay was carried out using a GST fusion protein containing the SH3 domain of PLCγ-1, in the absence (lane 1) or presence (lanes 2–6) of 1 mM of each of the five peptides. Binding of dynamin by the PLCγ-1 SH3 domain was assayed by blotting with antidynamin antibodies. As for the cortactin–SH3 domain-associated beads, peptides P2 and P1 inhibited dynamin binding, and some inhibition was demonstrated by the P4 peptide. d, Peptide sequence of the Dyn 2 COOH terminus. The four proline rich sequences are underlined. The bottom shows the sequences of the five synthetic peptides used to test the inhibition of SH3–dynamin interactions. The consensus sequence PXXPSRP was found to inhibit Dyn 2 binding to the GST–cortactin SH3 domain.

Mentions: Based on the interaction of dynamin with other proteins, it is likely that an association between dynamin and cortactin is mediated by a direct physical interaction of the SH3 domain of cortactin with the proline-rich COOH terminus of dynamin. To test this, a recombinant GST-fusion protein containing the cortactin SH3 domain was immobilized to glutathione-Sepharose and incubated with a cytosolic extract of NIH/3T3 cells. As shown in Fig. 2 a, the GST–cortactin SH3 domain-coupled beads bound significant amounts of dynamin, whereas GST beads alone did not. To define the specific peptide regions by which dynamin binds cortactin, synthetic peptides corresponding to individual polyproline sequences of the dynamin C-tail were included in the fibroblast homogenate before addition of the homogenate to the GST column. Two of the peptides used (P1 and P2) competed away the cortactin–dynamin interaction thereby preventing dynamin from binding to the beads whereas three other proline-rich peptides had no effect (Fig. 2 b). A sequence comparison of peptides P1 and P2 revealed the presence of a common motif, PxxPSRP (Fig. 2 d). The overlapping sequence P3, which lacks the proline at the COOH end of the consensus motif, did not compete for binding to dynamin. Because dynamin was shown previously to interact with the SH3 domain of PLCγ-1 (Scaife et al. 1994; Seedorf et al. 1994), the same peptides also were used to analyze the specificity of the interaction. As is the case with cortactin, the binding of PLCγ-1 to dynamin was also inhibited by peptides P1 and P2 (Fig. 2 c). In contrast, dynamin binding was significantly blocked by peptide P4 containing the sequence xxPxRP, which partially resembles the putative cortactin-binding consensus sequence. Thus, there is overlapping, yet nonidentical sequence specificity in the binding of the dynamin PRD to the SH3 domains of two distinct proteins.


Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW - J. Cell Biol. (2000)

The SH3 domain of cortactin binds dynamin. a, A cytosolic extract of NIH/3T3 cells was incubated with GST or GST–cortactin SH3 domain that was immobilized on glutathione Sepharose beads (lanes 1 and 2) or immunoprecipitated with the Dyn 2-specific antibody (lane 3). The bound proteins were fractionated and analyzed by blotting with an antidynamin mAb. As shown by the prominent dynamin band in lane 2, a substantial amount of dynamin was retained on the GST–cortactin SH3 domain beads as compared with the GST beads alone (lane 1). b, The GST pull-down assay was carried out in the absence or presence of one of the five synthetic peptides (P1–P5) at 0.1- or 1-mM concentrations. Binding of dynamin to the immobilized GST–cortactin SH3 domain was measured by blotting with an antidynamin mAb. Note that the P2 and P1 peptides were the most effective in blocking dynamin binding to the GST–SH3 domain beads (c). As a control the GST pull-down assay was carried out using a GST fusion protein containing the SH3 domain of PLCγ-1, in the absence (lane 1) or presence (lanes 2–6) of 1 mM of each of the five peptides. Binding of dynamin by the PLCγ-1 SH3 domain was assayed by blotting with antidynamin antibodies. As for the cortactin–SH3 domain-associated beads, peptides P2 and P1 inhibited dynamin binding, and some inhibition was demonstrated by the P4 peptide. d, Peptide sequence of the Dyn 2 COOH terminus. The four proline rich sequences are underlined. The bottom shows the sequences of the five synthetic peptides used to test the inhibition of SH3–dynamin interactions. The consensus sequence PXXPSRP was found to inhibit Dyn 2 binding to the GST–cortactin SH3 domain.
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Related In: Results  -  Collection

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Figure 2: The SH3 domain of cortactin binds dynamin. a, A cytosolic extract of NIH/3T3 cells was incubated with GST or GST–cortactin SH3 domain that was immobilized on glutathione Sepharose beads (lanes 1 and 2) or immunoprecipitated with the Dyn 2-specific antibody (lane 3). The bound proteins were fractionated and analyzed by blotting with an antidynamin mAb. As shown by the prominent dynamin band in lane 2, a substantial amount of dynamin was retained on the GST–cortactin SH3 domain beads as compared with the GST beads alone (lane 1). b, The GST pull-down assay was carried out in the absence or presence of one of the five synthetic peptides (P1–P5) at 0.1- or 1-mM concentrations. Binding of dynamin to the immobilized GST–cortactin SH3 domain was measured by blotting with an antidynamin mAb. Note that the P2 and P1 peptides were the most effective in blocking dynamin binding to the GST–SH3 domain beads (c). As a control the GST pull-down assay was carried out using a GST fusion protein containing the SH3 domain of PLCγ-1, in the absence (lane 1) or presence (lanes 2–6) of 1 mM of each of the five peptides. Binding of dynamin by the PLCγ-1 SH3 domain was assayed by blotting with antidynamin antibodies. As for the cortactin–SH3 domain-associated beads, peptides P2 and P1 inhibited dynamin binding, and some inhibition was demonstrated by the P4 peptide. d, Peptide sequence of the Dyn 2 COOH terminus. The four proline rich sequences are underlined. The bottom shows the sequences of the five synthetic peptides used to test the inhibition of SH3–dynamin interactions. The consensus sequence PXXPSRP was found to inhibit Dyn 2 binding to the GST–cortactin SH3 domain.
Mentions: Based on the interaction of dynamin with other proteins, it is likely that an association between dynamin and cortactin is mediated by a direct physical interaction of the SH3 domain of cortactin with the proline-rich COOH terminus of dynamin. To test this, a recombinant GST-fusion protein containing the cortactin SH3 domain was immobilized to glutathione-Sepharose and incubated with a cytosolic extract of NIH/3T3 cells. As shown in Fig. 2 a, the GST–cortactin SH3 domain-coupled beads bound significant amounts of dynamin, whereas GST beads alone did not. To define the specific peptide regions by which dynamin binds cortactin, synthetic peptides corresponding to individual polyproline sequences of the dynamin C-tail were included in the fibroblast homogenate before addition of the homogenate to the GST column. Two of the peptides used (P1 and P2) competed away the cortactin–dynamin interaction thereby preventing dynamin from binding to the beads whereas three other proline-rich peptides had no effect (Fig. 2 b). A sequence comparison of peptides P1 and P2 revealed the presence of a common motif, PxxPSRP (Fig. 2 d). The overlapping sequence P3, which lacks the proline at the COOH end of the consensus motif, did not compete for binding to dynamin. Because dynamin was shown previously to interact with the SH3 domain of PLCγ-1 (Scaife et al. 1994; Seedorf et al. 1994), the same peptides also were used to analyze the specificity of the interaction. As is the case with cortactin, the binding of PLCγ-1 to dynamin was also inhibited by peptides P1 and P2 (Fig. 2 c). In contrast, dynamin binding was significantly blocked by peptide P4 containing the sequence xxPxRP, which partially resembles the putative cortactin-binding consensus sequence. Thus, there is overlapping, yet nonidentical sequence specificity in the binding of the dynamin PRD to the SH3 domains of two distinct proteins.

Bottom Line: Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia.Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle.These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

Show MeSH
Related in: MedlinePlus