Dynamin 2 Mutations Implicated in Charcot-Marie-Tooth Disease

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Title: Dynamin 2 Mutations Implicated in Charcot-Marie-Tooth Disease
Author: Tassin, Tara Charisse
Abstract: Dynamins are large (100 kDa ) GTPases responsible for severing the necks of nascent vesicles during clathrin - and caveolae -mediated endocytosis , and are implicated in a variety of other cellular processes , including macropinocytosis , phagocytosis , and cytoskeletal organization . Mammalian cells contain three dynamin genes , encoding dynamin 1 (expressed in neurons and neuroendocrine cells ) , dynamin 2 (ubiquitously expressed ) , and dynamin 3 (enriched in testes , but also found in pre - and post -synaptic regions of neurons ) . Dynamin 2 was identified as a locus for Charcot -Marie -Tooth disease (CMT ) and Centronuclear Myopathy (CNM ) . CMT is a peripheral neuropathy affecting 1 in every 2 ,500 people , making it one of the most commonly inherited neurological disorders . CNM causes progressive loss of muscle tone without primary neuronal involvement . In this study , the effects of two CMT mutations were characterized in order to gain insight into the causes of the disease . The two mutations , K558E and delDEE (a deletion of residues 551 -553 ) , are both located in the Pleckstrin Homology (PH ) domain (approximately residues 520 -630 ) , which mediates the binding of dynamins to phosphoinositide lipids and to βγ subunits of heterotrimeric G proteins . The overall goal of the project was to determine how the mutations influence fundamental properties of dynamin 2 , including : 1 . Self -assembly and concentration -dependent GTPase activation ; 2 . Binding to phosphatidylinositol - (4 ,5 ) - bisphosphate (hereafter termed PIP2 ) and stimulation of GTPase activity by PIP2 ; 3 . Stimulus -dependent tyrosine phosphorylation ; and 4 . Interaction with G -βγ . In summary , I have found that Dyn 2 -K558E undergoes normal self -assembly and self -activation , but that its activation by PIP2 -containing vesicles is drastically reduced . Consistent with this observation , the ability of the isolated K558E PH domain to bind to PIP2 -containing vesicles was also impaired . Because full -length Dyn 2 -delDEE could not be expressed in Sf9 cells , I was unable to determine effects of this deletion on its self -assembly , self -activation , or activation by PIP2 vesicles . However , I took advantage of the bacterially -expressed GST -tagged PH domain to demonstrate that deletion of residues DEE does not affect binding to PIP2 , whereas it strongly (6 -20 fold ) enhances the interaction with G -βγ . This enhanced binding may be significant in explaining the role of the delDEE mutation in CMT disease , as previous studies have shown that G -βγ inhibits the GTPase activity of dynamin 1 . Although full -length Dyn 2 -delDEE protein could not be obtained for in vitro analysis , I was able to express the full -length mutant in mammalian cells , allowing me to examine its ability to undergo tyrosine phosphorylation . Consistently , Dyn 2 -delDEE underwent approximately 2 -3 fold higher levels of tyrosine phosphorylation than either wild -type dynamin 2 or Dyn 2 -K558E in Src -expressing cells stimulated by EGF or isoproterenol . Mutation of the two tyrosines (individually or in combination ) previously shown to be the major Src -phosphorylated sites in dynamins significantly reduced tyrosine phosphorylation in both wild -type and mutant dynamins . Finally , I compared the effects of overexpression of wild -type dynamin 2 and Dyn 2 -delDEE on stimulus -dependent activation of the MAP kinases Erk1 /2 . These experiments were motivated by earlier studies indicating that maximal Erk activation cannot occur if receptor -mediated endocytosis is inhibited . Overexpression of Dyn 2 -delDEE reduced Erk activation by 70 % , and activation was further reduced by mutation of the two phosphorylatable tyrosines . Mutation of the phosphorylatable tyrosines in wild -type dynamin 2 resulted in a 50 % inhibition of Erk activation . Overall , the results of my analysis demonstrate that two CMT mutations within the same domain of dynamin 2 have distinctly different properties . Future studies will be aimed at determining if these mutants impair endocytosis by distinct mechanisms .
URI: http : / /hdl .handle .net /2152 .5 /738
Date: 2010-05-14

Citation

Dynamin 2 Mutations Implicated in Charcot-Marie-Tooth Disease. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /738 .

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