Focus: Protein dephosphorylation
Dephosphorylation of Proteins with Shrimp Alkaline Phosphatase
Shrimp Alkaline Phosphatase (Cat.# M8201) can be used to dephosphorylate proteins in cell lysates. This activity can be used as a tool to study the phosphorylation state of proteins. Treated and untreated lysates can be analyzed by SDS-PAGE followed by Western blot analysis to detect differences in migration between phosphorylated and dephosphorylated proteins. This article shows an example of this technique used to examine the possible phosphorylation of two corepressors, N-CoR and SMRT by MEKK1.
From the
article Jonas, B.A. and Privalsky, M.L. (2004) J. Biol. Chem.
279, 54676–86.
Section of Microbiology, Division of Biological Sciences, University
of California, Davis, California 95616
Introduction
Shrimp Alkaline Phosphatase (SAP) and Calf Intestinal Alkaline Phosphatase (CIAP) are commonly used in molecular biology to dephosphorylate the 5′ phosphorylated ends of DNA or RNA. Both SAP and CIAP have been used to dephosphorylate proteins as well (1). This article shows an example of using SAP as a tool to identify the phosphorylation state of proteins.
The paralogs N-CoR and SMRT are corepressors of transcription. These proteins have a related structure and amino acid sequence and interact with many of the same transcription factors to repress transcription, yet the cellular function and regulation of these proteins is different. Jonas and Privalsky explored the distinct kinase signaling pathways regulating SMRT and N-CoR. In an experiment using SAP, they showed that the expression of MEKK1, a protein involved in the MAPK cascade, alters the migration of SMRT on a gel due to phosphorylation while N-CoR is unaffected, suggesting that SMRT is phosphorylated in response to MEKK1 while N-CoR is not.
Methods
CV-1 cells (1.5 × 105 cells/well in a 6-well plate) were transfected with the appropriate mammalian expression vectors using Effectene® transfection reagent (Qiagen). Cells were collected 48 hours after transfection by mechanical scraping and lysed by a 30-minute incubation at 4°C in 250µl of cell extraction buffer containing 25mM HEPES (pH 7.8), 300mM NaCl, 1.5mM MgCl2, 1% Triton® X-100, 0.1mM dithiothreitol, 0.2mM phenylmethylsulfonyl fluoride, and 1X complete protease inhibitor cocktail (GMD Biosciences, Inc.). Lysates were clarified by centrifugation at 14,000rpm for 30 minutes at 4°C, and divided into two equal aliquots. One aliquot was treated for 30 minutes at 37°C with 10 units of Shrimp Alkaline Phosphatase (Cat.# M8201), and one aliquot was mock-treated. The samples were then resolved by SDS-PAGE using the Novex® NuPAGE® Tris-acetate 3–8% gradient gel system. The electrophoretograms were visualized by immunoblotting using rabbit polyclonal antibody directed against either SMRT (diluted 1:2,000; Affinity Bioreagents) or N-CoR (diluted 1:500; Upstate Biotechnology, Inc.), horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (diluted 1:3,000, Bio-Rad), and the ECL™ Plus Western blot detection system (Amersham Biosciences). The chemiluminescent signals were captured and quantified using the Fluorchem® 8900 digital detection system.
Full-length SMRT and N-CoR (>2,400 amino acids long) were too large to accurately detect a change in the electrophoretic mobility due to phosphorylation/dephosphorylation. The constructs used in this experiment contained the Gal4 DNA Binding Domain fused to either the C-terminal 699 amino acids for SMRT (Gal4DBD-SMRT) or the C-terminal 508 amino acids for N-CoR (Gal4DBD-N-CoR). This C-terminal SMRT construct was sufficient to confer inhibition of SMRT by MEKK1 in two-hybrid assays.
Results and Conclusions
The upper panel of Figure 1 shows that the mobility of the SMRT construct was decreased by the cotransfection of an active MEKK1 allele (lane 4). This reduced mobility was reversed by incubation with SAP (lane 5). In contrast, MEKK1 had little or no effect on an equivalent N-CoR construct (lower panel). SAP treatment increased the mobility of N-CoR regardless of MEKK1 activity (lower panel, lanes 2 and 5), suggesting that N-CoR is constitutively phosphorylated. These data suggest that SMRT is more extensively phosphorylated in response to MEKK1 activity than is N-CoR.
Figure 1. MEKK1 detectably alters the electrophoretic mobility of a SMRT construct but not that of an N-CoR construct. Gal4DBD-SMRT (upper panel) and Gal4DBD-N-CoR (lower panel) constructs were transfected into CV-1 cells. The cells were also cotransfected with a constitutively active allele of MEKK1 as indicated. Control cells not expressing the corepressor constructs were analyzed in lane 1. The cells were lysed; aliquots of each lysate were incubated (lanes 2 and 5) or not (lanes 1, 3, and 4) with shrimp alkaline phosphatase (SAP); and the lysates were analyzed by SDS-PAGE and immunoblotting with either anti-SMRT or anti-N-CoR antiserum. White dashed lines indicate the positions of the corepressor in the absence of MEKK1 and in the absence of shrimp alkaline phosphatase treatment. WB, Western blot. Reprinted with the kind permission of Dr. Martin L. Privalsky, University of California, Davis and The Journal of Biological Chemistry.
The authors note that the effects of phosphorylation on electrophoretic mobility can be difficult to interpret. Electrophoretic mobility can be affected by numerous factors including the phosphorylation site and its chemical environment. These phosphatases also have substrate specificities that have not been fully explored. The utility of SAP or CIAP for dephosphorylation of proteins may need to be empirically determined for each situation.
Reference
- Fontao, L. et al. (2003) Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus. Mol. Cell. Biol. 14, 1978–92.
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