IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with ␤-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating ␣-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. Cell-cell adhesion is dynamically rearranged in various situations including the establishment of epithelial cell polarity, compaction of early embryogenesis, wound healing, cell scattering, and tumorigenesis (for reviews see Refs. 1-3). Cadherin is a well known calcium-dependent cell-cell adhesion molecule. The cytoplasmic domain of cadherin binds to ␤-catenin, and this complex is linked to the actin cytoskeleton by ␣-catenin. It is well known that this linkage is essential for the cadherinmediated cell-cell adhesion (for a review see Ref. 4). Therefore, it is likely that dynamic rearrangement of the cadherin-catenin complex is crucial for the above phenomena. However, little is known about the regulatory mechanism underlying the rearrangement of the cadherin-catenin complex.Cdc42 and Rac1, members of the Rho family, participate in the regulation of actin reorganization (for reviews see Refs. 5 and 6). Recent studies have suggested that they are required for maintaining the cadherin-mediated cell-cell adhesion (7-11). Target molecules for Cdc42 and Rac1 have been identified to be p21-activated kinase (12-14), WASP 1 (15, 16), IQGAP1 (17-19), and IQGAP2 (20), those for Cdc42 to be N-WASP (21) and MRCK-␣, -␤ (22), and those for Rac1 to be Sra1 (23), POR1 (24), and POSH (25). However, the mechanism underlying the regulation of cadherin-mediated cell-cell adhesion by Cdc42 and Rac1 has been unknown.We have recently found that IQGAP1 regulates the cadherinmediated cell-cell adhesion (10). IQGAP1 interacts with ␤-catenin and E-cadherin both in vitro and in vivo. The overexpression of IQGAP1 induces the dissociation of ␣-catenin from the cadherin-catenin complex and results in reduction of the Ecadherin-mediated cell adhesive activity in EL cells but not in L cells expressing E-cadherin mutant in which the cytoplasmic domain is deleted and replaced by the carboxyl-terminal half of ␣-catenin (nE␣CL cells) (26). The inhibitory effect of IQGAP1 on the E-cadherin-mediated cell-cell adhesion is counteracted by the coexpression of dominant active Cdc42 (Cdc42 Val12 ). Thus, IQGAP1 together with Cdc42 and Rac1 appear to regulate the cell-cell adhesion through the rearrangement of the cadherin-catenin complex. However, how Cdc42 and Rac1 regulate the IQGAP1 function remains to be clarified.In the present study, we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. We found that IQGAP1 bound to the amino terminus of ␤-catenin and thereby dissociated ␣-catenin from the ␤-catenin-␣-catenin complex. GTP␥S⅐GST-

Intramembrane proteolysis by presenilin-dependent ␥-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-␤. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val3Gly and the Lys3Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 A␤-like peptide (N␤)) should be produced. Here we showed that N␤ species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel N␤, N␤21 and C-terminally elongated N␤25, which were produced in an ϳ5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated A␤. PS pathogenic mutants increased the production of the longer species of A␤ (A␤42) from ␤-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of N␤. Strikingly, chemicals that modify the A␤42 level caused parallel changes in the N␤25 level. These results demonstrated that the characteristics of C-terminal elongation of N␤ and A␤ are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of A␤-or N␤-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for A␤42 production.