Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Abstract

Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved phosphoserine/phosphothreonine-specific protein phosphatase and member of the PPP family of phosphatases. Calcineurin is unique, however, in its activation by Ca 2+ and calmodulin. This ubiquitously expressed phosphatase controls Ca 2+ -dependent processes in all human tissues, but is best known for driving the adaptive immune response by dephosphorylating the nuclear factor of the activated T-cells (NFAT) family of transcription factors. Therefore, calcineurin inhibitors, FK506 (tacrolimus), and cyclosporin A serve as immunosuppressants. We describe some of the adverse effects associated with calcineurin inhibitors that result from inhibition of calcineurin in nonimmune tissues, illustrating the many functions of this enzyme that have yet to be elucidated. In fact, calcineurin has essential roles beyond the immune system, from yeast to humans, but since its discovery more than 30 years ago, only a small number of direct calcineurin substrates have been shown (∼75 proteins). This is because of limitations in current methods for identification of phosphatase substrates. Here we discuss recent insights into mechanisms of calcineurin activation and substrate recognition that have been critical in the development of novel approaches for identifying its targets systematically. Rather than comprehensively reviewing known functions of calcineurin, we highlight new approaches to substrate identification for this critical regulator that may reveal molecular mechanisms underlying toxicities caused by calcineurin inhibitor-based immunosuppression.

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

“…To further expand the set of CN-binding motifs, we next developed in silico tools, based on SLiMs from our ProP-PD selections and previously published CN-binding sequences (Table S4), to discover PxIxIT and LxVP sequences in the human proteome. Validated CN-binding sequences were used to construct specificity-determinant models that reflect the physiochemical preferences of the core and flanking residues of each motif ( Figures 3A and B; Table S2) (Roy and Cyert, 2019;Sheftic et al, 2016) (Nguyen et al bioRxiv 306779). Positionspecific Scoring Matrices (PSSMs), statistical models that include weighted sequence information at each motif position, were built to represent these specificity determinants.…”

“…Consequently, CN inhibitors FK506 (Tacrolimus) and cyclosporin A (CsA), are commonly prescribed as immunosuppressants (Liu, 1993). Unfortunately, due to CN inhibition in non-immune tissues, these drugs also cause a range of unwanted effects, including hypertension, diabetes, and seizures (Roy and Cyert, 2019). Systematically mapping CN signaling pathways throughout the body will aid in understanding and potentially ameliorating these effects.…”

“…The CN heterodimer, composed of two subunits (catalytic: CNA, regulatory: CNB), recognizes substrates by binding to PxIxIT and LxVP SLiMs ( Figure 1A), a mechanism that is evolutionarily conserved ). However, positioning of these motifs with respect to each other, and to sites of dephosphorylation varies between substrates, and these motifs play distinct roles during dephosphorylation (Nygren and Scott, 2016;Roy and Cyert, 2019). Under non-signaling conditions, the CN heterodimer is inactive due to masking of the active site by autoinhibitory sequences in CNA.…”

“…Under non-signaling conditions, the CN heterodimer is inactive due to masking of the active site by autoinhibitory sequences in CNA. During signaling, Ca 2+ binding sites in CNB become occupied, and Ca 2+ -bound calmodulin interacts with CNA to affect conformational changes that disrupt autoinhibition and activate the enzyme (Roy and Cyert, 2019). PxIxIT docking on CNA is unaffected by CN activation, thus these peptides target CN to substrates and regulators under both basal and signaling conditions.…”

“…PxIxIT docking on CNA is unaffected by CN activation, thus these peptides target CN to substrates and regulators under both basal and signaling conditions. In contrast, LxVP motifs bind to a hydrophobic groove that becomes accessible only after calmodulin binds to CNA, and are thought to orient phosphorylated residues for catalysis (Roy and Cyert, 2019). Mutation of PxIxIT or LxVP sequences in substrates or their conserved binding surfaces on CN disrupts dephosphorylation, and many CN inhibitors, including CsA, FK506, and the viral A238L protein, function by blocking SLiM binding (Grigoriu et al, 2013).…”