Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin.
Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.
Vaughan
P S
PS
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-0369, USA. vaughan4@nd.edu
Leszyk
J D
JD
Vaughan
K T
KT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D000072159:Dynactin Complex; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D004398:Dyneins