A Novel and Conserved Protein-Protein Interaction Domain of Mammalian Lin-2/CASK Binds and Recruits SAP97 to the Lateral Surface of Epithelia

Abstract

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice.

We thank Morgan Sheng for the Myc-tagged SAP97 construct and Ken Cadigan for helpful discussion. We also thank Robert Kruger and the laboratory of Jack Dixon for his help in maintaining a large-scale fly cage and collecting embryos.

Seonok Lee was supported by NIH Genetics training grant (5-T32 GM07544). Ben Margolis is an investigator of the Howard Hughes Medical Institute. This work was supported in part by NIDDK grant (2P50DK39255). We are also grateful to Thomas Komorowski, manager of the University of Michigan Diabetes Research Center Morphology and Image Analysis Core (NIH grant 5-P60-DK20572), for his assistance with confocal microscopy.

S. Lee and S. Fan made approximately equal contributions to this work.