FIG. 3.

S50A mutant m-calpain resistance to ERK-induced activation and activity in low and high calcium concentrations. (A) The activity of in vitro-transcribed and -translated wild-type and mutant (S50A) m-calpains was measured against that of recombinant m-calpain (Calbiochem) to determine their ability to cleave the calpain substrate Tau (0.5 μg). Addition of ERK to the in vitro-translated wild type, the T381A mutant m-calpain, and recombinant m-calpain showed equivalent abilities to cleave Tau within 10 min, as shown by loss of the Tau band at 65 kDa. On the other hand, the S50A mutant m-calpain exhibits a significant reduction in ERK activation, as shown by reduced loss of Tau (n = 3). Of the m-calpains tested none were shown to be able to cleave Tau alone (NT) or in the presence of 2 μM calcium (Ca⁺⁺) during a 10-min incubation. (B) Recombinant m-calpain is activated by 2 μM calcium over time. Early time points do not demonstrate noticeable levels of Tau cleavage; however, by 60 min all Tau is cleaved. Wild-type in vitro-translated m-calpain demonstrated a similar time course of calcium-induced activity (not shown). In vitro-translated mutant S50A m-calpain was also activated by calcium, but only at a 100-fold-higher level (200 μM); at 2 μM calcium no loss of Tau was noted over the 60-min time period (data not shown) (n = 3).