P nitrophenyl phosphate

To test how the presence of phosphatase affects the phosphorylation status of CaMKII when the kinase is active, increasing amounts of λ-phosphatase (200–800 units, New England Biolabs) and 1 mM MnCl₂ was added to samples of glass-immobilized CaMKII, in the presence of activation buffer containing 5 μM Ca²⁺/CaM. 1 unit of λ-phosphatase is defined as the amount of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate in 1 min at 30°C (New England Biolabs). After 45 min of incubation, the flow chambers were washed with 2 mL DPBS to remove the λ-phosphatase and the activation buffer. The phosphorylation of Thr 286 and Thr 305/306 was then examined using the immunofluorescence assay described above. No further reduction in phosphorylation was observed upon adding more than 400 units of λ-phosphatase and this is considered as a saturating amount of phosphatase.

SapM activity was assayed as described previously (Saleh and Belisle, 2000 (link); Zulauf et al., 2018 (link)). In a 96 well plate, 3 µg of CFP protein was diluted with water and added to 10X buffer (1M Tris base pH 6.8) with 20 mM Sodium tartrate to reduce background phosphatase activity, and 50 mM p-nitrophenyl phosphate (pNPP) for a total volume of 200 µL (New England Biolabs, Ipswich, MA). Tartrate is an inhibitor of some phosphatases, but SapM activity is unaffected by tartrate (Saleh and Belisle, 2000 (link)). Despite the addition of tartrate, the phosphatase assay used is not specific for SapM. The residual activity in the ∆secA2 and ∆satS mutants can be attributed to SatS-independent phosphatases. The plate was incubated at 37°C in a plate reader, and the absorbance at 405 nm was measured every three minutes for four hours. Over the linear portion of the kinetic assay, we calculated the rate of pNPP conversion by calculating the slope of the line generated by plotting Abs_{405 nm} as a function of time. These slopes were then normalized to the WT rate of change, which we set to 100%.