Our studies extend the genetic description of C. elegans
LIN-2 to its potential role in vertebrate epithelial cells, and provide several new insights about its possible function. We demonstrate that CASK is ubiquitously expressed and is localized predominantly to either the basal, lateral or basolateral plasma membrane domains in different epithelial cell types. In addition, we show that hCASK binds to the heparan sulfate proteoglycan syndecan-2 via its PDZ domain, and to the actin/spectrin-binding protein 4.1, presumably through a previously characterized motif located between the SH3 and GUK domains (Marfatia et al., 1995
; Lue et al., 1994
). Based on these associations, we speculate that in epithelial cells CASK mediates a direct link between the extracellular matrix and the cortical actin cytoskeleton through syndecan and protein 4.1, respectively (Fig. ). These interactions may have functional significance for membrane signaling and assembly of cell polarity.
Figure 8 A hypothetical model of hCASK mediating a direct link between extracellular matrix and actin cytoskeleton by binding to syndecan and protein 4.1. hCASK binds to syndecan via its PDZ domain, and to protein 4.1 presumably through a sequence motif located (more ...)
Human CASK demonstrates a high degree of overall identity with rat CASK, Drosophila
CAMGUK, and C. elegans
LIN-2. It is composed of an NH2
-terminal CAMK, a PDZ domain, an SH3 domain, a protein 4.1-binding motif, and a domain homologous to GUK. Sequence alignments with the alpha subunit of rat brain CAMKII (Lin et al., 1987
) suggest that hCASK is likely to bind calmodulin, but is unlikely to be an active enzyme. The latter conclusion is based on substitution within hCASK of several residues that are conserved among all enzymatically active CAMKs, including two within the putative ATP binding site (Colbran and Soderling, 1990
; Hanks et al., 1988
). Hata et al. (1996)
have shown that recombinant fragments of CASK containing its putative calmodulin-binding site bind calmodulin efficiently in a calcium-dependent manner. However, they found no evidence for CAMK activity using recombinant proteins, and did not observe autophosphorylation of CAMK domains expressed in bacteria, mammalian cells, or insect cells (Hata et al., 1996
). In native CAMKII, calmodulin binding releases autoinhibition on the catalytic domain (reviewed in Braun and Schulman, 1995
). By analogy, calmodulin binding may regulate CASK by inducing a conformational change, although in this case it would not be expected to induce kinase activity.
Sequence comparisons of hCASK with porcine and yeast GUK enzymes suggest that hCASK has the potential to be an active guanylate kinase enzyme, although this activity has not been tested (Zschocke et al., 1993
; Stehle and Schulz, 1992
). p55 is the only other MAGUK that conserves or conservatively substitutes all of the residues known to be essential for GUK enzymatic activity. Active GUK domains might contribute to signaling by affecting guanine nucleotide levels and activity of small GTP-binding proteins, or by possessing effector function themselves when bound to guanine nucleotides. Recently, a novel protein family called GKAP (guanylate kinase–associated protein) or SAPAP (SAP90/PSD-95–associated protein) has been shown to bind to the GUK domain of PSD-95/ SAP90 and related synaptic proteins (Kim et al., 1997
; Takeuchi et al., 1997
), although their sequences are novel and functions unknown. None of these newly identified proteins bind the GUK domain of CASK, leaving open the possibility that additional GKAP/SAPAP-like proteins remain to be discovered.
In this study we demonstrate that CASK is ubiquitously expressed and shows distinct localization patterns predominantly at the basal, lateral, or basolateral membrane domains in different epithelial cell types. CASK appears to be excluded from the apical membrane compartment. Despite its apparently distinct localization patterns, CASK is always associated with a membrane domain that faces bloodborne growth factors and hormones. This fact suggests a role for CASK analogous to that of C. elegans LIN-2, which is thought to localize to the basal membrane domain of vulval precursor epithelial cells in association with the LET-23 growth factor receptor.
To identify a binding partner for hCASK in polarized epithelial cells, we performed a yeast two-hybrid screen of a human liver library using the PDZ domain as bait. We found that the hCASK PDZ domain binds to the transmembrane protein syndecan-2. X-ray crystallography and mutational analysis had previously demonstrated that the COOH-terminal residues of the cytoplasmic tail of PDZ-binding transmembrane proteins confer the specificity of the PDZ domain-tail interaction (Doyle et al., 1996
; Kim et al., 1995
). Using an oriented peptide library technique, Songyang et al. (1997)
assigned PDZ domains into classes according to their peptide-binding specificities. hCASK, like p55 and Tiam-1, falls in the so-called class II, selecting peptides with hydrophobic or aromatic side chains at position –2 relative to the carboxy terminus. The physical basis for this specificity is consistent with the recent x-ray crystalographic structure of the hCASK PDZ domain (Daniels et al., 1998
). Inspection of known PDZ-binding motifs reveals that E at position –3 is very common (Doyle et al., 1996
). Syndecan-2, which terminates in the sequence EFYA, as well as neurexin, which ends with residues EYYV, are both predicted to bind to the PDZ domain of CASK. It is possible that CASK has other PDZ-binding partners in addition to syndecan and neurexin, that terminate with hydrophobic or aromatic residues.
In vitro binding studies reported here demonstrate that the PDZ domain of CASK binds specifically to the COOH-terminal EFYA motif present in both syndecans 1 and 2. The apparent Ki in our binding assay with syndecan peptides consisting of the terminal eight residues is in the range of 40–70 μM, consistent with previously reported PDZ interactions (Songyang et al., 1996). Although we were unable to coimmunoprecipitate a complex of CASK and syndecan from native cells, this result has been the experience for other PDZ interactions for which there is either genetic or functional proof of an interaction.
In addition to these in vitro binding studies, we demonstrate that CASK and syndecans 1 and 2 share the same membrane domain–restricted distribution in different epithelial cells. Although we lack antibodies to investigate a possible colocalization with syndecans 3 and 4, the COOH-terminal sequence identity among all four syndecan gene products suggests that CASK can interact with all syndecans. This possibility is of interest because while all contain a highly conserved cytoplasmic domain, syndecans have divergent extracellular domains, cellular expression patterns, and presumed functions (Carey, 1997
). For example, syndecan-1, expressed almost exclusively in epithelial cells of mature tissue, is thought to act as a matrix receptor, regulate cell morphology, and participate in a coreceptor complex with bFGF receptor. In contrast, syndecan-4, expressed by both epithelial and fibroblastic cells, is enriched and codistributed with integrins in focal contacts, raising the possibility that syndecan-4 forms a coreceptor complex with integrin receptors (Woods and Couchman, 1994
In this study we show that CASK, like p55 and hDlg, binds to the NH2
-terminal 30-kD domain of erythrocyte protein 4.1. This domain is highly conserved among 4.1- related proteins, raising the possibility that MAGUK proteins can bind to other 4.1 family members such as ezrin, radixin, moesin, merlin, or talin. All of these proteins have been implicated in organizing actin in the cortical cytoskeleton (reviewed in Tsukita and Yonemura, 1997
). Perhaps an interaction with MAGUK proteins offers a mechanism for tethering transmembrane proteins into the cortical actin network. This connection would serve to localize proteins to the proper cell membrane domain and bring them into proximity with other proteins in the same signaling pathway. For example, CASK could stabilize syndecan at the basolateral surface where it binds extracellular matrix and acts as a coreceptor for ligands such as bFGF. CASK might then recruit other proteins used in signaling events downstream of the bFGF receptor. Although direct interactions between CASK and signaling proteins have not yet been demonstrated, other MAGUKs clearly have such associations. For example, hDlg binds via its proline-rich NH2
-terminal domain to the SH3 domain of the nonreceptor tyrosine kinase p56 lck
in lymphocytes (Hanada et al., 1997
). It also binds the tumor suppressor APC in epithelial cells and neurons (Matsumine et al., 1996
). The synaptic proteins PSD-95 and PSD-93 bind neuronal nitric oxide synthase (Brenman et al., 1996a
and Brenman et al., 1996b
). Finally, the tight junction MAGUK ZO-1 binds through its SH3 domain to a novel serine threonine kinase, ZO-1–associated kinase, or ZAK (Balda et al., 1996
The syndecan–CASK–protein 4.1 interaction may explain the results of previous studies demonstrating a regulated association between syndecan and the actin cytoskeleton. In stably transfected Schwann cells, syndecan-1 has been seen to colocalize transiently with actin filaments during cell spreading. Similarly, antibody-mediated clustering of syndecan molecules on the cell surface also induces actin colocalization (reviewed in Carey, 1997
). Mutational analysis has implicated the involvement of a tyrosine residue within the syndecan-1 cytoplasmic domain in this connection (Carey et al., 1996
); however, the potential role of CASK as a link has not yet been explored.
Our results suggest several novel roles for CASK in epithelial cells. Activation of the bFGF receptor tyrosine kinase is optimal when the bFGF ligand is presented as a ternary complex bound to the heparan sulfate moieties of syndecan (Rapraeger et al., 1991
; Yayon et al., 1991
). If CASK is involved in bFGF signaling it may in this context facilitate receptor tyrosine kinase (RTK) function by restricting syndecan to the basolateral cell membrane with the receptor. A previously published study supports a role for CASK in proper syndecan localization (Miettinen et al., 1994
). When wild-type syndecan was transfected into Madin-Darby canine kidney cells, it appeared on the basal and lateral membrane domains. A truncated syndecan missing the last twelve amino acids of the cytoplasmic tail mislocalized to both the apical and basolateral cell surfaces. These data may be explained by the inability of truncated syndecan to bind the PDZ domain of CASK. CASK might be necessary for syndecan targeting to the correct membrane domains; alternatively, CASK may be stabilizing syndecan at the membrane by linking it to the actin cytoskeleton.
In vertebrate epithelial cells and in nematode vulval precursor cells, indirect evidence suggests that CASK and LIN-2, respectively, are each in close proximity to a receptor tyrosine kinase. LIN-2 is responsible for localizing the LET-23 receptor to the basal cell surface through a mechanism that does not involve direct binding to the RTK (Simske et al., 1996
). There is genetic evidence in C. elegans
for involvement of other proteins in intracellular LET-23 binding (Simske et al., 1996
). However, by analogy to the proposed CASK–syndecan–bFGFR interaction in mammalian cells, it is possible that LIN-2 binds to the C. elegans
syndecan homolog, perhaps forming a coreceptor complex with the LET-23 RTK. Interestingly, C. elegans
syndecan terminates with the same four residues as the vertebrate syndecan EFYA (Carey, 1997
), suggesting that the LIN-2 PDZ domain is capable of binding to its COOH-terminal tail. Whatever the mechanism of association between CASK/LIN-2 and the cytoplasmic domain of RTKs, this proximity raises the possibility of a role in signal transduction. For example, such a role might be to recruit other signaling molecules, to act itself as a substrate for the RTK, or to regulate the availability of other substrates. There is presently no direct evidence for this model; however, CASK/LIN-2 has several domains that might serve such purposes. These hypotheses provide directions for future experiments concerning the role of CASK in vertebrate epithelial cell signaling.