P301L TAU DEMONSTRATES ALTERED MODULATION OF CALMODULIN-DEPENDENT PDE1 AND CALCINEURIN IN VITRO

Microtubule binding protein Tau is highly expressed in neurons with either 3 or 4 microtubule binding domains (MBD, 3R and 4R respectively). Its primary function is to limit the depolymerization of axonal tubulin structures thereby adding to the stabilization of neurons. When this process is impaired by mutations that alter the ratio of 3R/4R Tau or affect Tau protein structure, Tau can gain pathological functions that are associated with Tau tangle formation and neuronal death. P301L Tau is one such example identified in families presenting with Frontotemporal dementia and Parkinson disease (e.g. Hutton et al., 1998; Dumanchin et al., 1998; Spillantini et al., 1998). The P to L mutation is in the second microtubule binding domain and imparts decreased affinity for binding microtubules (Fischer et al., 2007). This same region is juxtaposed to the calmodulin binding domain in Tau (Avila et al., 1990) such that the mutation could also affect Tau interaction with this central regulator of calcium signal transduction. Recombinant 441-Tau, 352-Tau and P301L-Tau were purified on RP-HPLC and then compared for effects on the activation of the calmodulin-dependent enzymes 3’,5’phosphodiesterase 1 (PDE1) and calcineurin (PP2b, PP3) in vitro. Increasing concentrations of calcium or Tau protein were employed and hydrolysis of cAMP to 5’AMP was monitored for PDE1 activity and release of phosphate from calcineurin substrate peptide (Anaspec 29962) was measured in calcineurin assays. Tau enhanced the activation of PDE1 by lowering the calcium concentration required to maximally activate the enzyme. A rank order for activation across Tau forms of P301L-Tau> 441-Tau> 352-Tau was observed. Similar comparisons were carried out with Tau and calcineurin where Tau demonstrated enzyme inhibition, with P301L-Tau being modestly less effective than wild-type Tau. Tau is capable of both positive and negative modulation of calmodulin-dependent enzymes in vitro, and the structural change caused by the P to L substitution in the MBD impacted the modulation. These results continue to highlight the microtubule binding domain in Tau as an important structure that can gain pathological properties including altered interactions with key cell signaling components involved in neuronal homeostasis.