Functional Analysis of the Nucleotide Binding Domain of Membrane-associated Guanylate Kinases

Olav Olsen,David S Bredt

doi:10.1074/jbc.m210165200

Abstract

Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.

Highlights

Tissue development, differentiation, and physiology require specialized cellular adhesion and signal transduction at sites of cell-cell contact
We conclude that the SH3GK domains of postsynaptic density-95 (PSD-95) and CASK do not bind GMP with affinities Ͻ1 mM
This study shows that the guanylate kinase (GK) domains of Membrane-associated guanylate kinases (MAGUKs) proteins PSD-95 and CASK do not bind to GMP with detectable affinity

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—DNA sequences encoding YGK (residues 2–187), rat PSD-95 (SH3GK, residues 417–724; GK, residues 532–711), and rat CASK (SH3GK, residues 591–909) were amplified by PCR and cloned in-frame into a His6-tagged expression vector [16]. Escherichia coli strain BL21 (DE3) (Stratagene) expressing PSD-95 and YGK constructs were grown in LB to an OD600 ϭ 0.8, induced with 100 ␮M isopropyl-1-thio-␤-D-galactopyranoside for 3 h, harvested by centrifugation, and sonicated in Buffer A (50 mM NaPO4 (pH 8.0), 300 mM NaCl, 10% glycerol). Following incubation with bacterial lysates, resin was extensively washed with 50 mM NaPO4 (pH 6.5), 300 mM NaCl, 10% glycerol, and subsequently with Buffer A containing 5 and 15 mM imidazole. Prior to the interaction assays, His6-tagged proteins, COS cell lysate, and coupled GST proteins were incubated in the presence or absence of 5 mM GMP for 1 h at 4 °C. Coupled GST fusion proteins were incubated with 20 ␮g of His6-tagged fusion proteins or 80 ␮l of COS cell lysate for 40 min Ϯ 5 mM GMP at 4 °C and extensively washed with either Buffer A or TEEN Ϯ 5 mM GMP. Retained proteins were eluted with SDS protein loading buffer, separated by PAGE, and analyzed by immunoblotting

RESULTS

Specific activity

DISCUSSION