Calcium Stimulates Self-Assembly of Protein Kinase C α <i>In Vitro</i>

<div><p>Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium <i>in vitro</i>. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC—mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P₂, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca²⁺ is minimally required and sufficient for clustering.</p></div

A C1a-C2 complex is minimally sufficient for self-assembly.

<p>A.) Schematic for the C1-C2 SPASM biosensors. B.) The C1a and C2 but not C1b and C2 biosensors forms punctae on glass coverslips in the presence of free calcium. The response is abrogated with pretreatment of the biosensor with TEV protease, where neither the N-termini nor C-termini of the biosensor forms punctae. Data from 6 fields of view per condition. C.) Analytical size exclusion chromatography of C1a - 10 nm—C2 in an EGTA buffered (left) or 300 μM free calcium (right) mobile phase. In both conditions the protein was run with or without a pre-incubation with TEV protease. Fluorescence of mCit and mCer in the eluant were assessed. Elution volume of the indicated peptides are compared against molecular standards (black dots) fit to a single exponential decay function (black line). The non-proteolyzed peptide did not come off the column in the presence of calcium. D.) HeteroFRET displayed as the ratio of mCit fluorescence intensity of mCer fluorescence intensity of the indicated biosensors, each at 50 nM in a solution buffered by EGTA. E.) HeteroFRET of C1a - 10 nm—C2 (WT intact or TEV proteolyzed) or D246N (100 nM) sequentially treated free calcium and EGTA. F.) The change in heteroFRET following addition of free calcium for C1a - 10 nm—C2 for the indicated concentration of biosensor. The data are fit to Hill binding model with a hill coefficient of 2.39 ± 0.09 (best fit and standard error; black line). G.) C1a-C2 biosensors with 3 lengths of ER/K linkers are assessed for punctae formation on glass coverslips all in the presence of 300 μM free calcium, data from ≥ 10 fields of view per condition. The longer length linkers systematically reduced punctae formation (middle) with no systematic effect on the relative intensity of punctae (right). H.) Normalized Δ heteroFRET and spot number for C1a –C2 with the indicated ER/K linker length, all fixed at 100 nM. I.) Model demonstrating an intramolecular complex between C1a and C2 is required and sufficient for self-assembly in the presence of free calcium.</p

PKCα clusters on liposomes through two independent mechanisms, one driven by DAG and one by PI(4,5)P₂.

<p>A—D) Steady-state anisotropy of PKCα-mCit (A, B) C2-mCit (C) and PKCα K197A/K199A-mCit (D) under the indicated conditions. All liposomes were matched with an 80:1 molar ratio of PS:PKCα where PS is 10% molar composition of liposome. Box and whisker represent min, max, mean, 25 and 75 percentiles of N ≥ 8 independent experiments. B.) Specific kinase activity of PKCα-mCit under otherwise matched conditions is plotted against steady-state anisotropy in the presence of free calcium. Shown is mean and SEM of N ≥ 6. All protein at 100 nM.</p

Regulatory domains self-assemble.

<p>Mean DLS autocorrelation data before (red) or after addition of free calcium (green) of full-length PKCα (left), the regulatory domains (middle), and C2 domain (right) at matched time points (2 min incubation) and concentration. Normalized particle mass is quantified at the far right. Box and whisker plots represent min, max, 25 and 75 percentile and median of N > 5. Each particle mass increased significantly following calcium addition (p>0.0001).</p

PKC forms distinguishable supramolecular states with different combinations of inputs.

<p>Using two experimental criteria PKC can be parsed into three unique supramolecular states. FRET puts a physical constraint on the proximity of PKC molecules within ~1.6 of <i>R</i>_<i>o</i> (mCer-mCit <i>R</i>_<i>o</i> = 5.4 nm; 8.6 nm) suggestive of direct interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.ref038" target="_blank">38</a>], but is sensitive to the orientation between chromophore dipoles such that the absence of FRET does not rule out an interaction. Calcium alone causes self-assembly of PKC which can be directly observed using biophysical approaches (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g001" target="_blank">Fig 1</a>), is not detectable by FRET (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g004" target="_blank">Fig 4</a>), and requires both the C2 and C1a domain (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g003" target="_blank">3</a>). Calcium plus liposomes containing DAG result in intermolecular FRET between PKC–mCit (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g004" target="_blank">Fig 4</a>), but require the full-length protein and not just the C2 domain (presumably requires C1 domains which are only known DAG binding sites; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g006" target="_blank">Fig 6</a>). Calcium plus liposomes containing PI(4,5)P₂ result in intermolecular FRET and the C2 domain is sufficient for this result (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.g006" target="_blank">Fig 6</a>). Schematic is organized to parallel schematics initially describing supramolecular structures proposed by John-Marie Lehn [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#pone.0162331.ref039" target="_blank">39</a>].</p

HomoFRET detects PKCα clustering on liposomes but not in solution.

<p>Fluorescence anisotropy can be used to monitor homoFRET. A characteristic feature of homoFRET in fluorescent proteins is a depolarization of fluorescence on the time scale of energy migration (10⁻¹⁰–10⁻⁹ seconds) in addition to depolarization occurring from rotational diffusion (tau > 10⁻⁸ seconds). The two effects on depolarization can be distinguished with time resolved anisotropy measurements (TCSPC or DWR). A.) Representative time-correlated single photon counting (TCSPC) data of mCit—PKCα-FLAG, PKCα-mCit-FLAG and PKCα-mCit-FLAG plus liposomes with and without free calcium. B.) Steady-state fluorescence anisotropy of mCit in C1a-SPASM-C2, PKCα-mCit and PKCα-mCit + liposomes as the protein is sequentially treated with excess EGTA, free calcium and EGTA (N = 16 for steady state measurements). The purple diamonds are simulated steady-state anisotropy values derived from a two-exponential fit of direct waveform recording (DWR) time-resolved anisotropy measurements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162331#sec002" target="_blank">methods</a>). The unilaminar liposome contained 88%PC:10%PS:2%DAG (molar %; PS to PKC molar ratio 32:1) for A and B. C.) Time course of mCit steady-state fluorescence anisotropy from C1a –SPASM—C2 following injection of free calcium (40 s) and EGTA (1200 s) (black line) or buffer blanks (red line). Shown in mean and standard deviation, N = 4 independent matched time course.</p

Calcium induces reversible self-assembly of PKCα <i>in vitro</i>.

<p>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).</p