Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain.
Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potently stimulate its GTPase activity. Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to define its functional domains. Limited digestion with papain split the protein into an approximately 7- to 9-kDa microtubule-binding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end of the polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.
Herskovits
J S
JS
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Shpetner
H S
HS
Burgess
C C
CC
Vallee
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D017124:Conserved Sequence; D034281:Dynamins; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D007074:Immunoglobulin G; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010206:Papain; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D007074:Immunoglobulin G; D010446:Peptide Fragments; D010455:Peptides; D014404:Tubulin; D016392:Proto-Oncogene Proteins pp60(c-src); D010206:Papain; D020558:GTP Phosphohydrolases; D034281:Dynamins