In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.

Abstract. Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla/0 and functionally rescues the flalO phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino-terminal motor domain, a central a-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP-PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla/0-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and tall. Furthermore, flalO-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA/0 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.

SUMMARY Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MEK inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.

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The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins. INTRODUCTIONTight junctions (TJ) are a hallmark of polarized epithelial cells, providing the paracellular barrier required to separate tissue spaces (Van Itallie and Anderson, 2004), contributing to maintenance of apical-basolateral cell polarity and providing a site for cell-cell signaling (Schneeberger and Lynch, 2004;Matter et al., 2005;Shin et al., 2006). At the ultrastructural level, tight junctions appear as highly organized strands that encircle the apical-lateral boundary. These strands are composed of transmembrane proteins such as occludin, tricellulin, and one or more members of the claudin family, which create the paracellular barrier. These proteins are, in turn, associated with a cytosolic plaque of proteins that is closely associated with the cortical cytoskeleton. The sequence of events that bring about the proper continuous apical localization of TJ proteins remains unknown. One approach to this problem has been to define components in upstream pathways required to induce cell polarity (Shin et al., 2006). Another approach has been to elaborate the biochemical interactions among the more than 40 transmembrane and cytosolic components of the TJ and to define their functional role in somatic cells using gene ablation, transcriptional inhibition, or dominant negative strategies (Schneeberger et al., 2004). Despite considerable efforts by many groups, the precise mechanism of junction assembly is poorly understood.The cytoplasmic protein ZO-1 is proposed to be one of the key regulators of TJ assembly (reviewed in Fanning, 2006). ZO-1 is member of a large family of membrane-associated scaffolding and signaling molecules known as the membrane-associated guanylate kinase homologues (MAGUKs). These proteins are characterized by a core motif of conserved protein-binding domains including one or more PDZ domains, an SH3 domain and a GUK domain (Funke et al., 2005). By analogy to other MAGUKs,...