Alternative splicing generates protein isoforms with distinct regulatory and functional properties, yet the biological consequences of many splice variants remain poorly understood. The cytoskeletal scaffold protein ADD3 exists in two major isoforms, including a long isoform (ADD3-L) containing exon 14 that has been associated with metastatic cancer phenotypes. Given the established regulation of ADD3 by calcium–calmodulin signaling, we developed a probabilistic framework to investigate how calcium dynamics influence the activation behavior of ADD3 isoforms. The model integrates stochastic Ca²⁺–calmodulin encounters, Gamma-distributed activation kinetics, and Markov state transitions to characterize calcium-dependent activation and localization patterns. Simulations suggest that inclusion of exon 14 may enhance activation persistence and reduce stochastic variability, particularly in calcium-rich environments. Extending the framework to spatial calcium gradients predicts preferential activation near membrane-proximal regions and a calcium-dependent redistribution of ADD3-L toward cytoplasmic and nuclear compartments. These findings indicate that calcium-enriched tumor microenvironments may stabilize ADD3-L activity and promote cytoskeletal plasticity associated with metastatic behavior. More broadly, this study demonstrates how probabilistic modeling can be used to link alternative splicing events to context-dependent cellular phenotypes and generate mechanistic hypotheses for experimental validation.

Co-authors: Abdullah Kahraman