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

Abstract

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.

Figure 1

Association of α4 with Mid1 and PP2A. (a) Yeast two-hybrid analysis showed that strains expressing Gal4-DBD-α4 grew on triple-drop out media (trp-, leu-, his-) only when also expressing PP2A, PP6, or Mid1 (residues 58–180), indicative of an interaction with α4. Controls included single transformants and PP1C that did not support growth, because it does not bind α4. The plate was scanned on a densitometer to show growth of yeast colonies. (b) COS7 cells were transfected with FLAG-tagged Mid1(58–180) with or without myc-tagged α4. Cells were lysed and immunoprecipitation was performed by using anti-FLAG M2 affinity gel. FLAG-Mid1(58–180) and α4 were detected in the immunoprecipitates by immunoblotting with anti-FLAG and anti-α4 antibodies. When present, myc-α4 was detected by anti-myc immunoblotting. (c) COS7 cells transfected with vector alone, FLAG-α4 (full-length), FLAG-α4(1–249), FLAG-α4(220–340), or FLAG-α4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed. FLAG-α4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG, anti-Mid1, and anti-PP2A-C immunoblotting.

Figure 2

Codependent distribution of GFP-Mid1 and FLAG-α4. (a) COS7 cells were fixed and stained with anti-α4 antibodies to show the distribution of the endogenous α4 protein in a nontransfected cell (first frame) and in a cell expressing GFP-Mid1 (second frame). The distribution of GFP-Mid1 itself is shown in green, and colocalization of GFP-Mid1 and endogenous α4 is shown in yellow in the merged image (fourth frame). (b) COS7 cells were cotransfected to express both GFP-Mid1 and FLAG-α4. Cells were fixed and GFP-Mid was visualized by direct fluorescence microscopy, and FLAG-α4 was stained as described. From multiple experiments, representative cells with increasing levels of FLAG-α4 (columns 1, 2, and 3, left to right) were compared for localization of FLAG-α4 and GFP-Mid1. In the merged images, nuclei were stained by DAPI. (c) Cells expressing both GFP-Mid1 and FLAG-α4 to give foci of GFP-Mid1 (green) were fixed and stained for β-tubulin (red) and DAPI (blue).

Figure 3

OS mutants of GFP-Mid1 associate with FLAG-α4. (a) COS7 cells were transfected to express GFP-Mid1(1–480) together with each of the following FLAG constructs: FLAG empty vector, FLAG-α4, and FLAG-α4(220–340). Cells were fixed and GFP-Mid1 was visualized by direct fluorescence microscopy. FLAG-α4 was visualized by anti-α4 immunofluorescence. Nuclei of the cells were visualized by DAPI staining. (b) COS7 cells were cotransfected to express FLAG-α4 and different GFP-Mid1 fusion constructs: GFP alone (as control), GFP-Mid1, GFP-Mid1(1–480), GFP-Mid1(L626P), and GFP-Mid1(C266R). Immunoprecipitation was performed by using anti-FLAG M2 affinity gel and FLAG-α4; GFP-Mid1 fusion proteins and PP2A were detected by immunoblotting with anti-FLAG, anti-GFP, and anti-PP2A. The mutant GFP-Mid1 proteins all bound FLAG-α4.

Figure 4

Dephosphorylation of GFP-Mid1 induced by α4 and MAP kinase activity required for Mid1 association with microtubules. (a) COS7 cells were cotransfected to express GFP-Mid1 together with either FLAG-α4 or FLAG empty vector. Immunoprecipitation was performed by using anti-FLAG M2 affinity gel, and FLAG-α4 and GFP-Mid1 were detected by anti-FLAG and anti-GFP antibodies. (b) COS7 cells were transfected as described and radiolabeled with [³²P]orthophosphate for 90 min. GFP-Mid1 was immunoprecipitated with anti-GFP antibodies and was subjected to PhosphorImager analysis after SDS/PAGE. Yield of GFP-Mid1 in the immunoprecipitates was determined by anti-GFP immunoblotting. FLAG-α4 in the cell lysate was detected by anti-FLAG immunoblotting. (c) COS7 cells were transfected to express GFP-Mid1, an initial image was captured (GFP:Mid1), and then the cells were treated with 10 μM UO126 and images were captured at the times indicated.

Figure 5

Model for regulation of Mid1 association with microtubules by phosphorylation. The Mid1 protein is shown as two domains: a larger, elongated N-terminal domain and a smaller, circular C-terminal domain that is either truncated or contains mutations in OS (cross-hatched). Mid1 binds to other proteins into high M_r complexes in the cytoplasm (large circle) or can bind to microtubules (rods shown on left side). The binding to microtubules is affected by MAP kinase, which is inhibited by compounds UO126 and PD98059, and by binding to α4 (small, yellow circle) that associates with protein phosphatase catalytic subunits (labeled PP) that promote dephosphorylation of Mid1 and its release from microtubules.