Abstract. The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin iliamerit/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CIM4 mAbs showed that ezrin and radixin, as well as moesin, are associated with CIM4, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CIM4 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CIM4 and actin-based cytoskeletons.

Abstract. To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), /El-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at A J, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. CellBiol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.T I-IE adherens junction (AJ) ~ represents a specialized class of cell-to-cell and cell-to-substrate associations (14): the adhesion molecules are cadherins (6, 11, 46) and integrins (21, 38), respectively. This type of junction is characterized by the well-developed undercoat on the cytoplasmic surface of the junctional membrane through which actin filaments are tightly associated. AJ is commonly believed to be directly involved in tissue morphogenesis. This feature is, most likely, related to the tight association between AJ and the force-generating actin filament bundles. In addition to these mechanical properties of A J, the notion has become widespread that AJ plays crucial roles in the control of cell growth and tissue morphogenesis through transmembrane signaling (6,11,19,26,38,50). Recently, the information of the adhesion molecule-based signaling in AJ is gradually increasing; for example, specific types of calciumdependent protease(4) and protein kinase C (24) were immunofluorescently shown to be highly concentrated at the undercoat of the cell-to-substrate AJ, suggesting that the site-specific proteolysis and/or phosphorylation on serine/threonine residues might be important signals used in AJ. Abbreviations used in this paper:A J, adherens junctions; pAb, polyclonal antibody.Evidence has been accumulating that tyrosine phosphorylation is another important signal in AJ (10,15,17,18,27,29,31,36,45,50). The relationship between AJ and tyrosine phosphorylation was first illuminated by Rohrschneider (36) who showed th...

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Protein kinase C (PKC) family requires phosphorylation of itself to become competent for responding to second messengers. Much attention has been focused on elucidating the role of phosphorylation in PKC activity; however, it remains unknown where this modification takes place in the cells. This study examines whether anchoring protein is involved in the regulation of PKC phosphorylation. A certain population of PKC⑀ in rat brain extracts as well as that expressed in COS7 cells was associated with an endogenous anchoring protein CG-NAP (centrosome and Golgi localized PKN-associated protein). Pulse chase experiments revealed that the associated PKC⑀ was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non-or hypophosphorylated PKC⑀ directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKC⑀ did not. PKC⑀ mutant at a potential phosphorylation site of Thr-566 or Ser-729 to Ala, possessing almost no catalytic activity, was associated and co-localized with CG-NAP at Golgi/centrosome area. On the other hand, wild type and a phosphorylation-mimicking mutant at Thr-566 were mainly distributed in cytosol and represented second messenger-dependent catalytic activation. These results suggest that CG-NAP anchors hypophosphorylated PKC⑀ at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction. PKC1 family is a key player in the cellular responses mediated by the second messenger diacylglycerol and the phorbol ester tumor promoters (1). Although the allosteric control is an essential feature of its action as a signal transducer, it has been revealed that an additional two types of control are also important as follows: one is phosphorylation of kinases themselves (2, 3), and the other is specific subcellular localization mediated by anchoring proteins (4, 5). The PKC family has three conserved in vivo phosphorylation sites as follows: the activation loop site and two sites located C-terminal to the kinase domain (6, 7), with the exception of the atypical PKC isozymes having a Glu residue at the most carboxyl position. Modification of these sites by combination of trans-and autophosphorylation is required to gain catalytically competent conformation in order to respond to second messengers (8 -11). It has been found that the activation loop site of PKC as well as other kinases such as PKB and p70S6K is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) (12-15). Because PDK1 kinase activity is constitutive (16), activation loop phosphorylation may be controlled by localization and conformation of the substrates. For instance, phosphorylation of PKB is triggered by translocation to the membrane through binding its pleckstrin homology domain with phosphatidylinositol-(3,4,5)-triphosphate, causing a conformational change that exposes the activation loop phosphorylation site (17). It is likely that the PKC family is also recruited to the particular place through binding with lipid or with anc...

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