The calmodulin regulator protein, PEP-19, sensitizes ATP-induced Ca ²⁺ release

Background: PEP-19 modulates the kinetics of Ca²⁺ binding to CaM. Results: An acidic region in PEP-19 binds Ca²⁺ and is essential for both modulating Ca²⁺ binding to CaM and sensitizing cells to ATP-induced Ca²⁺ release. Conclusion: Simply binding to CaM is not sufficient to account for the biological activities of PEP-19. Significance: Regulating ligand-induced Ca²⁺ release gives PEP-19 the potential to broadly affect cell signaling. PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca ²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺release.We show that PEP-19 increases the sensitivity of HeLa cells to ATPinduced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding toCaMvia the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.