Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit alpha 4.
Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the alpha4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged alpha4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of alpha4. Localization of Mid1 and alpha4 was influenced by one another in transiently transfected cells. Mid1 could recruit alpha4 onto microtubules, and high levels of alpha4 could displace Mid1 into the cytosol. Metabolic (32)P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length alpha4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by alpha4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.
Liu
J
J
Center for Cell Signaling, University of Virginia School of Medicine, P.O. Box 800577, Charlottesville, VA 22908-0577, USA.
Prickett
T D
TD
Elliott
E
E
Meroni
G
G
Brautigan
D L
DL
D000818:Animals; D019556:COS Cells; D051379:Mice; D008868:Microtubule Proteins; D008870:Microtubules / Q000737:chemistry*; D009154:Mutation; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D021122:Protein Subunits; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D008868:Microtubule Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D014157:Transcription Factors; C109052:MID1 protein, human; D044767:Ubiquitin-Protein Ligases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2