HEPARIN BINDING OF THROMBOSPONDIN-1 IS REGULATED BY A NEWLY IDENTIFIED PKA PHOSPHORYLATION SITE

Abstract

Introduction: Thrombospondin-1 (TSP1) is a multifunctional glycoprotein involved in cell-to-cell and cell-to-matrix communication. Its N-terminal domain contains a well-defined, high-affinity heparin-binding region that mediates interactions with heparan sulphate proteoglycans and other extracellular ligands. These interactions influence a wide range of cellular processes, including cell adhesion, spreading, proliferation, and migration. Previous structural and biochemical studies have identified a cluster of positively charged residues (most notably R29, R42, R76/R77) that form the core of the TSP1 heparin-binding surface. Our workgroup has identified Ser44 as a protein kinase A (PKA) phosphorylation site in TSP1, located within the heparin-binding region, suggesting that phosphorylation at this position may modulate heparin affinity through local structural or electrostatic effects. Aim: The aim of this study was to investigate whether phosphorylation at Ser44 influences the ability of the TSP1 N-terminal domain to bind heparin. Material and methods: Site-directed mutagenesis was used to introduce substitutions at Ser44, generating Ser44Ala (phosphonull) and Ser44Asp (phosphomimetic) variants of TSP1. Recombinant TSP1 N-terminal fragments were then expressed in BL21(DE3) E. coli cells. For the heparin-binding assay, equal amounts of each recombinant protein were incubated with heparin-agarose beads. The input, unbound, and bound samples were analyzed by SDS-PAGE, followed by gel staining or Western blotting. Results: Sequencing confirmed the successful introduction of the Ser44Ala and Ser44Asp mutations. All recombinant TSP1 N-terminal proteins were successfully expressed at comparable levels. In the heparin-agarose assay, both the wild-type and the phosphonull mutant bound efficiently to heparin. In contrast, the phosphomimetic mutant exhibited markedly reduced binding. Conclusion: Our findings support the concept that phosphorylation of Ser44 plays a regulatory role in modulating heparin binding by the TSP1 N-terminal domain.