Protein Kinase D Modulation of Cardiac Protein Phophatases

Abstract

Protein Kinase D1 (PKD1) is an important stress stimuli transducer affecting myriad cellular functions. Despite a crucial role in pathological cardiac remodeling, few cardiac PKD1 targets have been identified. Recent studies suggest PKD1 could target protein phosphatases (PP) other than the well-known substrate slingshot. Here we test this by measuring phosphatase activity and expression in ventricular homogenates (n=10) from cardiac-specific PKD1 knockout mice (cKO) vs. their wildtype littermates (WT). Using the EnzChek phosphatase assay kit with the 6,8-difluoro-4-methylumbelliferyl phosphate substrate (DiFMUP), PP1, PP2a, PP2b and PP2c phosphatase activity was measured as tautomycin-, LB100-, calcineurin- and sanguinarine chloride-sensitive DiFMUP fluorescence respectively. PP1 activity was slightly higher in cKO vs. WT homogenates (1.4 ± 0.1 vs. 1.0 ± 0.1) at baseline. Inhibition of the other PKD isoforms with 25 nM CRT0066101, reduced PP1 activity in both WT and cKO (to 0.8 ± 0.4 and 0.7 ± 0.1 respectively). PP2a activity was slightly reduced in cKO homogenates (1 ± 0.2 vs. 0.7 ± 0.2) at baseline, and strongly reduced with CRT treatment in both WT and cKO (0.3 ± 0.1 vs. 0.2 ± 0.1 respectively). PP2b activity was unaltered in WT vs. cKO homogenates (1.0±0.2 vs. 1.0±0.1) but CRT strongly reduced PP2b activity (0.2±0.1 vs. 0.1±0.2 in WT and cKO respectively). PP2c activity was also similar at baseline and decreased following CRT inhibition (0.2 ± 0.1 vs. 0.1 ± 0.1 in WT and cKO). Interestingly PP2A protein expression was reduced in cKO vs. WT homogenates. Our results are a first step in defining PKD isoforms as regulators of cardiac protein phosphatase activity. Future experiments will be critical to uncover the molecular mechanisms that underlie the fine-tuning of phosphatase activity in health and disease by PKD signaling.