Human CASK/LIN-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane of epithelial cells

Abstract

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.

Figure 1

Comparative domain organization of hCASK-related proteins. Sequence homology of hCASK with rat CASK (Hata et al., 1996), C. elegans LIN-2 (Hoskins et al., 1995), human erythrocyte p55 (Ruff et al., 1991), and alpha subunit of rat brain CAMKII (Lin et al., 1987). hCASK protein contains a domain homologous to CAMKII, a conserved calmodulin-binding domain (CBD), a PDZ domain, an SH3 domain, a potential protein 4.1–binding motif (4.1), and a domain homologous to guanylate kinase (GUK). Percent amino acid identities within defined domains are noted in comparison with hCASK.

Figure 2

hCASK is ubiquitously expressed. Northern blot of human poly(A)⁺ mRNA probed with hCASK cDNA. The tissue source of the blotted RNA is indicated above each lane. Three differently sized transcripts are apparent at 8.5 kb, 4.4 kb, and 3.1 kb. All are large enough to encode full-length cDNA (2.765 kb). The position of size markers are indicated on the left (kb). Hybridization to 1.9-kb β-actin transcript is shown in the bottom panel as a loading control. Heart and skeletal muscle characteristically show a second β-actin mRNA form at 1.5 kb.

Figure 3

Anti-hCASK antibodies recognize one band at 112 kD, consistent with full-length cDNA and the nematode homolog. Immunoblot of rat tissues probed with affinity-purified anti-hCASK antibodies. Whole tissue samples were normalized by total protein, and were resolved by SDS-PAGE (10%). Positions of molecular weight markers are shown.

Figure 4

CASK localizes to distinct membrane domains in different epithelial cell types. Rat tissue sections were stained with affinity-purified anti-hCASK antibodies. (a) In choroid plexus epithelial cells, CASK localizes along the basal membrane; bar, 100 μm. (b) In colonic epithelial cells, CASK is enriched at the base of the lateral plasma membrane; bar, 40 μm.

Figure 6

CASK and syndecan-1 colocalize in situ in epithelial tissues. Mouse tissue sections were stained with affinity-purified rabbit anti-hCASK and monoclonal rat anti-syndecan-1 antibodies and visualized with Texas red–conjugated anti-rabbit and FITC-conjugated anti-rat secondary antibodies, respectively. (a, b) In hepatocytes, both CASK and syndecan-1 are located along the basal sinusoidal membrane. (c, d) In small intestinal enterocytes, the two proteins are localized at the lateral plasma membrane. CASK also stains a subset of the vessels that syndecan-1 stains in the lamina propria. (e, f) In the pancreas, CASK and syndecan-1 colocalize on both the basal and lateral membrane domains of acinar cells; bar, 40 μm.

Figure 5

The PDZ domain of hCASK binds to the COOH-terminal sequence of syndecans. (a) Alignment of the cytoplasmic domains of rat syndecan family members demonstrate the high degree of homology among the four proteins (reviewed in Bernfield et al., 1992). In particular, note the conservation of the last four residues (in bold), which in other proteins are known to determine PDZ tail-binding specificity. (b) Direct binding study with syn-2 peptide and hCASK PDZ fusion protein. Syn-2 peptide representing the last eight residues was coupled to CNBr- activated Sepharose beads. The beads with immobilized peptide were incubated with GST-PDZ recombinant fusion protein with increasing concentrations of syn-2 or syn-1 peptides (0–200 μM) in solution. Controls consisted of GST alone incubated with peptides immobilized on beads; GST-PDZ fusion protein with peptide-free Sepharose beads (Beads alone); and GST-PDZ fusion protein incubated with beads coupled to syn-2 peptide and 50 μM control dlg peptide in solution (Ctr). The dlg control peptide (KKKKETDV-COOH) has optimal binding affinity for murine Dlg PDZ domain (Songyang et al., 1997).

Figure 5

The PDZ domain of hCASK binds to the COOH-terminal sequence of syndecans. (a) Alignment of the cytoplasmic domains of rat syndecan family members demonstrate the high degree of homology among the four proteins (reviewed in Bernfield et al., 1992). In particular, note the conservation of the last four residues (in bold), which in other proteins are known to determine PDZ tail-binding specificity. (b) Direct binding study with syn-2 peptide and hCASK PDZ fusion protein. Syn-2 peptide representing the last eight residues was coupled to CNBr- activated Sepharose beads. The beads with immobilized peptide were incubated with GST-PDZ recombinant fusion protein with increasing concentrations of syn-2 or syn-1 peptides (0–200 μM) in solution. Controls consisted of GST alone incubated with peptides immobilized on beads; GST-PDZ fusion protein with peptide-free Sepharose beads (Beads alone); and GST-PDZ fusion protein incubated with beads coupled to syn-2 peptide and 50 μM control dlg peptide in solution (Ctr). The dlg control peptide (KKKKETDV-COOH) has optimal binding affinity for murine Dlg PDZ domain (Songyang et al., 1997).

Figure 7

hCASK binds to the actin/spectrin-binding protein 4.1. (a) [¹²⁵I]-labeled 30-kD fragment of protein 4.1 was incubated with GST-hCASK, GST-p55, and GST alone immobilized on nitrocellulose. Radiolabeled protein 4.1 bound GST-hCASK and GST-p55, but not GST alone. (Left) SDS-PAGE of expressed fusion proteins used for the experiment. (b)¹²⁵I-labeled 30-kD fragment of protein 4.1 was incubated with GST-hCASK and GST alone coupled to glutathione Sepharose beads. Significantly more radiolabeled protein 4.1 bound to GST-hCASK than to GST alone, and the binding was effectively competed by incubation with an excess of nonradiolabeled protein 4.1. Experiments were performed in triplicate.

Figure 7

hCASK binds to the actin/spectrin-binding protein 4.1. (a) [¹²⁵I]-labeled 30-kD fragment of protein 4.1 was incubated with GST-hCASK, GST-p55, and GST alone immobilized on nitrocellulose. Radiolabeled protein 4.1 bound GST-hCASK and GST-p55, but not GST alone. (Left) SDS-PAGE of expressed fusion proteins used for the experiment. (b)¹²⁵I-labeled 30-kD fragment of protein 4.1 was incubated with GST-hCASK and GST alone coupled to glutathione Sepharose beads. Significantly more radiolabeled protein 4.1 bound to GST-hCASK than to GST alone, and the binding was effectively competed by incubation with an excess of nonradiolabeled protein 4.1. Experiments were performed in triplicate.

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 between the SH3 and the GUK domains, as previously demonstrated for p55 and hDlg. Syndecans have been shown to bind extracellular matrix components and to form noncovalent coreceptor complexes with receptor tyrosine kinases, such as bFGFR. hCASK, in close proximity to the receptor tyrosine kinase, could act on the cytoplasmic surface to recruit proteins used in signaling events downstream of the bFGF receptor, or alternatively, could itself be a substrate for the receptor.