Calcium Stimulates Self-Assembly of Protein Kinase C α In Vitro
Fig 1
Calcium induces reversible self-assembly of PKCα in vitro.
A.) Recombinant PKCα-mCit-FLAG or PKCα-FLAG (300 nM) was differentially fractionated into soluble (S) and pellet (P) fractions following high speed centrifugation in two independently performed experiments. Fractionation occurred sequentially in EGTA buffered, free calcium (300 μM), and EGTA buffered solutions where the fractions circled were retained for the subsequent fractionation. Fractions were separated on SDS-PAGE and probed with mCit fluorescence (top) or an anti-PKCα antibody (bottom) in independent experiments. B.) Intensity autocorrelation of dynamic light scattering (DLS) of recombinant PKCα-FLAG (1 μM) sequentially diluted into buffers containing excess EGTA, free calcium, and EGTA with 15 min incubation between readings. The black line is a single exponential fit, and error bars are s.e.m. of 3 independent readings (left). Quantification of the ensemble particle mass normalized to the initial condition of indicated protein from DLS (Right; N ≥ 8, box-and-whisker represents min, max, 25 and 75 percentile and median). C.) Representative fluorescent images of recombinant PKCα-mCit-FLAG (200 nM) sequentially in indicated buffer non-specifically adhered to a glass coverslip. Samples were incubated in buffers for 10 minutes at 22°C before being adhered to slides. (left) Bright spots were identified when the ratio of mCit intensity deviates by >1.12 from the neighboring pixels. Data was quantified from 6 fields of view for each condition (right). D.) Differential sedimentation and spot number on coverslips were assessed as a function of free calcium concentration. The data are least squares fit to a single binding function (solid lines). Error bars represent standard deviation (N = 3 differential sedimentation and N = 5 microscopy). E.) Representative blot and quantification of differential fractionation of endogenous PKCα in 5x diluted and clarified HEK cell lysate (detergent free) probed with anti-PKCα antibody and corresponding silver stain (left) and quantified (Right; N = 4; min, max, 25 and 75 percentile and median).