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 develop ment at the midline. Gene mapping has identified OS mutations within a prot ein called Midi. Wild-type Midi predominantly colocalizes with microtubules , in contrast to mutant versions of Midi that appear clustered in the cytos ol. Using yeast two-hybrid screening, we found that the alpha4-subunit of p rotein phosphatases 2A/4/6 binds Midi. Epitope-tagged alpha4 coimmunoprecip itated endogenous or coexpressed Midi from COS7 cells, and this required on ly the conserved C-terminal region of alpha4. Localization of Midi and alph a4 was influenced by one another in transiently transfected cells. Midi cou ld recruit alpha4 onto microtubules, and high levels of alpha4 could displa ce Midi into the cytosol. Metabolic P-32 labeling of cells showed that Midi is a phosphoprotein, and coexpression of full-length a4 decreased Midi pho sphorylation, indicative of a functional interaction. Association of green fluorescent protein-Midi with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Midi associati on with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein p hosphatase that is targeted specifically to Midi by alpha4. Human birth def ects may result from environmental or genetic disruption of this regulatory cycle.