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Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin
P S Vaughan1, J D Leszyk, K T Vaughan
1Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-0369, USA. vaughan4@nd.edu
The Journal of Biological Chemistry
|May 8, 2001
Summary
Phosphorylation of cytoplasmic dynein intermediate chains regulates binding to dynactin. This study identifies serine 84 as the key phosphorylation site, impacting dynein-dynactin complex formation and intracellular transport.
Area of Science:
- Cell Biology
- Molecular Motors
- Protein Phosphorylation
Background:
- Dynactin acts as an adaptor for cytoplasmic dynein via intermediate chain (IC) and p150(Glued) interaction.
- The regulation of this dynein-dynactin interaction is not fully understood.
Purpose of the Study:
- To investigate if phosphorylation of the dynein intermediate chain regulates its interaction with dynactin.
- To identify the specific phosphorylation site and its functional consequences.
Main Methods:
- Two-dimensional gel electrophoresis and phosphatase treatment of cytoplasmic dynein.
- In vitro overlay assays with p150(Glued) and intermediate chains.
- Mass spectrometry to identify phosphorylation sites.
- Site-directed mutagenesis to create phosphomimetic and non-phosphorylatable mutants.
- In vitro binding assays and in vivo overexpression studies.
Main Results:
- Cytoplasmic dynein intermediate chains exist in at least two variants, one dephosphorylated by phosphatases.
- p150(Glued) binds to dephosphorylated intermediate chains but not phosphorylated ones.
- Mass spectrometry identified serine 84 on the intermediate chain as the phosphorylation site.
- Mutants mimicking dephosphorylation bound p150(Glued) and disrupted dynein transport, while phosphomimetic mutants showed reduced binding and no transport disruption.
Conclusions:
- Phosphorylation at serine 84 is a critical regulatory mechanism for the cytoplasmic dynein-intermediate chain interaction with dynactin.
- This phosphorylation event modulates the binding affinity and affects dynein-dependent intracellular transport.