Synergistic Activation of Dynamin GTPase by Grb2 and Phosphoinositides

Baryłko, Barbara; Binns, Derk D.; Lin, Keng Mean; Atkinson, Mark A.; Jameson, David M.; Yin, Helen L.; Albanesi, Joseph P.

doi:10.1074/jbc.273.6.3791

Cited by 120 publications

(95 citation statements)

References 46 publications

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“…Previous studies have shown that dynamin's basal GTPase activity can be stimulated by dynamin assembly induced by binding of polymers to its PRD (Warnock et al, 1997;Barylko et al, 1998). Thus, the differential effect of GST-SNX9 compared with GST-amph1, may reflect differential oligomerization of these proteins.…”

Section: Snx9 Stimulates Dynamin's Basal Gtpase Activitymentioning

confidence: 99%

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

Soulet

Yarar

Leonard

et al. 2005

MBoC

181

233

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Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domaincontaining proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity. INTRODUCTIONThe GTPase dynamin plays a critical role in clathrin-mediated endocytosis (CME; Hinshaw, 2000). Dynamin self-assembles into a collar-like structure around the necks of deeply invaginated clathrin-coated pits (CCPs) where it is believed to directly mediate membrane fission and vesicle release. Dynamin is also associated with newly formed coated pits (Damke et al., 1994;Evergren et al., 2004) where it may function to regulate early stages of coated pit maturation (Sever et al., 2000a;Song and Schmid, 2003;Song et al., 2004).Although only one isoform of dynamin exists in Drosophila and in Caenorhabditis elegans, mammals express at least three isoforms, which are ϳ70% identical to each other. Dynamin-1, the first identified protein in the dynamin family, is expressed exclusively in neuronal cells where it functions in synaptic vesicle recycling (Shpetner and Vallee, 1989;Nakata et al., 1991;Sontag et al., 1994). Dynamin-2 is ubiquitously expressed (Cook et al., 1994;Sontag et al., 1994) and localizes to endocytic CCPs where it functions, like dynamin-1, in endocytosis (Damke et al., 1994;Altschuler et al., 1998). However, dynamin-2 may also be involved in vesicle formation at the Golgi Kreitzer et al., 2000), in regulating actin dynamics (Schafer, 2004), in cell signaling (Kranenburg et al., 1999;Fish et al., 2000), and has recently been localized to the centriole (Thompson et al., 2004). Dynamin-3, which is most highly expressed in testis but also detectable in neurons (Gray et al., 2003) and in lung (Nakata et al., 1993), has been less well studied.Dynamins are atypical GTPases, distinguished by their large size, low affinity for GTP, and high intrinsic rates of GTP hydrolysis (Sever et al., 2000b;Song and Schmid, 2003). Moreover, dynamin can self-assemble in solution (Hinshaw and Schmid, 1995) or on liposome templates into rings and spiral-like structures (Stowell et al., 199...

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Section: Snx9 Stimulates Dynamin's Basal Gtpase Activitymentioning

confidence: 99%

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

Soulet

Yarar

Leonard

et al. 2005

MBoC

181

233

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“…Assembly of dynamins is facilitated by their binding to negatively charged scaffolds, including PI(4,5)P 2 -containing vesicles (15,(25)(26)(27). These scaffolds reduce the concentrations of dynamin required for polymerization and GTPase activation and stimulate GTPase activities to higher levels than can be achieved by dynamin alone.…”

Section: Stabilization Of Dnm2 Polymers By the R369w Mutation-mentioning

confidence: 99%

Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Wang

Baryłko

Byers

et al. 2010

Journal of Biological Chemistry

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Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNMassociated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis. Autosomal dominant CNM2 is a congenital disorder that commonly results in muscle weakness and wasting, ptosis, and ophthalmoplegia (reviewed in Ref. 1). As the name implies, the most evident histopathological feature is the presence of a large number of muscle fibers of centrally (rather than peripherally) located nuclei. Other characteristics include a relative increase in the number of type I fibers, hypertrophy of these fibers, and the presence of sarcoplasmic strands distributed radially around the central nuclei. In 2005, Bitoun et al. (2) reported the identification of four mutations in the DNM2 (dynamin 2) gene in patients with autosomal dominant CNM, and an additional seven mutations have since been identified (3, 4). DNM2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles into helical polymers around the necks of vesiculating membranes, thereby providing force for their constriction and scission (reviewed in Refs. 5-8). In addition to its well characterized roles in endocytosis and Golgi budding, DNM2 is also implicated in regulation of the actin (9, 10) and microtubule (11, 12) cytoskeletons. The DNM2 molecule consists of five functional domains: an N-terminal catalytic domain; a so-called "middle domain" implicated in dynamin-dynamin interactions; a PH domain involved in phosphoinositide binding; a GTPase effector domain, which interacts with the catalytic domain and stimulates its GTPase activity; and a C-terminal proline/arginine-rich domain, which mediates interactions of dynamin with other proteins. Most of the currently kn...

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“…Phosphoinositides, PI(4,5)P 2 in particular, bind clathrin adaptors such as AP-2 (14-16), AP180 (17,18), and epsin (19,20), as well as the GTPase dynamin (21,22), which is critically required for the fission of endocytic vesicles (23,24). They also regulate a variety of proteins that control actin nucleation (13,25,26).…”

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confidence: 99%

Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

Kim

Chang

Daniell

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

112

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Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exo͞endocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool. N eurotransmission and its reliability depend on the efficient and precise recycling of synaptic vesicles in the presynaptic nerve terminal (1, 2). Vesicle fusion and neurotransmitter release are followed by rapid retrieval and repackaging of synaptic vesicle membrane components into new functional vesicles. Morphologic, biochemical, genetic, and functional evidence points to an important role of the clathrin-mediated recycling pathway in this process (1,(3)(4)(5). Alternative and parallel pathways may also participate in recycling (1, 2, 6-9).Growing evidence suggests that phosphoinositides play an important role in the synaptic vesicle cycle via their interaction with endocytic and actin regulatory proteins (10-13). Phosphoinositides, PI(4,5)P 2 in particular, bind clathrin adaptors such as AP-2 (14-16), AP180 (17,18), and epsin (19,20), as well as the GTPase dynamin (21, 22), which is critically required for the fission of endocytic vesicles (23, 24). They also regulate a variety of proteins that control actin nucleation (13,25,26). Actin, in turn, is thought to play an important role in clathrin-mediated recycling by generating and organizing cytoskeletal scaffolds at endocytic zones (27-29) and, possibly, by contributing to the translocation of newly formed endocytic vesicles to the synaptic vesicle cluster (30-34).Synaptojanin 1 is a polyphosphoinositide phosphatase highly enriched in presynaptic nerve terminals and localized to recycling intermediates in synaptosomes (35,36). Its COOHterminal proline-rich domain interacts with several accessory factors implicated in synaptic vesicle recycling, including amphiphysin (35), endophilin (37), (38), intersectin (34, 39), and syndapin͞pacsin (40, 41). Its NH 2 -terminal Sac1-like module hydrolyzes the phosphates of PI(3)P, PI(4)P, and PI(3,5)P 2 (42), whereas its central phosphatase module hydrolyzes the 5Ј phosphate of PI(4,5)P 2 and PI(3,4,5)P 3 (43,44). By means of these enzymatic activities, synaptojanin can dephosphorylate polyphosphoinositides to phosphatidylinositol and thereby function as a negative regulator of the interactions between membranes, coat proteins, and regulatory factors for actin.In mice knockout studies, the absence of ...

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Synergistic Activation of Dynamin GTPase by Grb2 and Phosphoinositides

Cited by 120 publications

References 46 publications

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

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