Systems-level analyses of phosphorylation-based signaling networks has transformed our understanding of kinase function, but knowledge of phosphatase signaling has lagged behind, primarily because global approaches to identify phosphatase substrates are lacking. Calcineurin, the conserved Ca2+/calmodulin-dependent protein phosphatase, is ubiquitously expressed and critically regulates Ca2+-dependent processes in the immune system, heart, and brain. However, in the literature only 27 substrates are attributed to calcineurin. By using both experimental and computational methods, we developed a novel pipeline to identify CN substrates. We have validated new substrates in CN in novel pathways and cellular regions.
Calcineurin is tightly controlled by Ca2+ and calmodulin, which activate the enzyme by relieving auto-inhibition of the active site, and revealing a critical binding pocket for "LxVP" substrate motifs. We are studying regulation of a conserved splice variant of the human CNAβ gene, CNAβ1, which promotes cardiac regeneration in vivo. The CNAβ1 C-terminus contains an LxVP sequence, which we showed auto-inhibits phosphatase activity by blocking substrate engagement. CNAβ1 has distinct enzymatic properties, and is relatively independent of calmodulin. Functional studies are identifying and characterizing unique protein partners for CNAβ1 and its substrates in cardiovascular and endocrine signaling.
In vivo, rapid regulation of weak, transient protein-protein interactions is essential for dynamically shaping cellular responses. Most of these occur via binding of a globular domain to a short (3-10 amino acids long) peptide motif or SLiM (Short Linear Motif), found primarily in disordered protein regions. The low affinities of SLiMs for their receptor domain, sequence degeneracy, and the prevalence of PTMs render SLiMs difficult to identify using current techniques. This project applies a technology recently developed by the Fordyce lab, use of spectrally encoded bead libraries, to analysis of SLiMs that bind to calcineurin, the conserved Ca2+/calmodulin dependent protein phosphatase and target of immunosuppressants (FK506 and Cys A).