MARCKS Redistribution Assay

• Designed to assay compounds for their ability to modulate PKC signaling
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The protein MARCKS (myristoylated alanine-rich C kinase substrate) is a ubiquitous substrate of PKC. MARCKS is a cytoskeletal protein and has been implicated in regulation of cell spreading, adhesion, phagocytosis, and membrane trafficking. MARCKS is localized at the plasma membrane with the N-terminal myristate inserted into the lipid bilayer, but upon activation by PKC it translocates to the cytosol.

The MARCKS Redistribution assay has been developed using histamine as reference agonist. Histamine activates endogenous H1 G-coupled receptors, which leads to Ca²⁺ release, PKC activation and ultimately MARCKS translocation from membrane to cytoplasm. Knockdown of endogenous H1 receptors abolishes the response to histamine.

The assay has been developed both in agonist and antagonist format. In the agonist format, histamine is used as reference agonist. In the antagonist assay, the broad PKC inhibitor staurosporine is used as the reference compound and several additional PKC inhibitors have been tested for activity in this assay. GF 109203X, Gö6983, and Ro 31-8220 (all broad isoform specificity) are able to antagonize MARCKS translocation in response of histamine, whereas Gö6976 (conventional PKC specific) is not.

Figure 1. Translocation of MARCKS-EGFP in response to histamine. Cells were treated with 100 μM histamine for 2 min (middle panel) in the presence (right panel) or absence (DMSO control, left panel) of the antagonist staurosporine. The MARCKS-EGFP translocation from the plasma membrane to perinuclear spots is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the MARCKS assay: A) Histamine concentration response in the MARCKS agonist assay (n=16). The EC50 is ~1 μM. Concentration response was measured in 9 point half log dilution series. Cells were treated with histamine for 2 min. Cells were then fixed and MARCKS translocation was measured using the Cellomics ArrayScan® V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (100 μM histamine) and negative control (0.25% DMSO). B) Staurosporine concentration response in the MARCKS assay run in antagonist format (n=16). Cells were incubated for 2 min with a half log dilution series of staurosporine in the presence of 100 μM histamine. Cells were then fixed and and analyzed on the Cellomics ArrayScan® V^TI Reader. % activity was calculated relative to the positive (1 μM staurosporine) and negative control (0.25% DMSO).The EC₅₀ of staurosporine is ~90 nM. C) Concentration response to a panel of PKC inhibitors in the MARCKS antagonist Redistribution assay (n=4). Cells were incubated for 2 min with a half log dilution series of the PKC inhibitors in the presence of 100 μM histamine. Cells were then fixed and and analyzed on the Cellomics ArrayScan® V^TI Reader. % activity was calculated relative to the positive (1 μM staurosporine) and negative control (0.25% DMSO).