Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated K D value was 3.3 ؋ 10 ؊7 M. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P 2 . PtdIns(4,5)P 2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P 2 . The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

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Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrinradixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKAinduced phosphorylation. In vitro [␥-33 P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growthsuppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.

Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr 477 as the primary target residue. We generated a pTyr 477 -phosphospecific antibody, which confirmed that Tyr 477 becomes phosphorylated in cells in a Src-dependent manner. Tyr 477 phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr 477 phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/ POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

Intercellular adhesion molecule-2 (ICAM-2) functions as a ligand for lymphocyte function-associated antigen-1 (LFA-1) and is involved in leukocyte adhesion. We studied intracellular associations of ICAM-2 using a peptide encompassing the cytoplasmic amino acids 231-254 as an affinity matrix. Among the proteins from placental lysates that bound to the peptide was alpha-actinin as demonstrated by immunoblotting. Purified, 125I-labeled alpha-actinin also bound to the peptide. Confocal microscopic analysis of Eahy926 cells demonstrated a colocalization of ICAM-2 and alpha-actinin. Of overlapping octapeptides covering the entire ICAM-2 cytoplasmic amino acids, ICAM-2241-248 bound alpha-actinin most avidly and effectively competed with the longer cytoplasmic peptide for binding. The site of interaction in alpha-actinin was studied using bacterially expressed alpha-actinin fusion proteins. Several constructs covering nonoverlapping regions of alpha-actinin bound to the ICAM-2 cytoplasmic peptide suggesting that multiple regions in alpha-actinin can mediate the interaction. These results, together with previously demonstrated interactions between alpha-actinin and the adhesion proteins ICAM-1, L-selectin, beta1- and beta2-integrins emphasize the role of alpha-actinin as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton.

We evaluated the role of Lck tyrosine kinase, an early e¡ector of T cell activation, in regulation of the membrane^cytoskeleton linker protein ezrin. Ezrin was constitutively tyrosine phosphorylated in wild-type and CD45-de¢cient Jurkat T cells, but not in Lck-de¢cient cells. However, phosphorylation was evident in cells, in which Lck activity had been restored by transfection. Phosphorylation was reduced by the Src family kinase inhibitor PP2 and increased by the tyrosine phosphatase inhibitor pervanadate, implying continuous tyrosine phosphorylation and dephosphorylation. Lck phosphorylated ezrin in vitro, and the major phosphotyrosine was identi¢ed as Y145. These results identify ezrin as the ¢rst cytoskeletal substrate for Lck. ß