Multi-PK antibodies: Powerful analytical tools to explore the protein kinase world

Abstract

Diverse biological events are regulated through protein phosphorylation mediated by protein kinases. Some of these protein kinases are known to be involved in the pathogenesis of various diseases. Although 518 protein kinase genes were identified in the human genome, it remains unclear how many and what kind of protein kinases are expressed and activated in cells and tissues under varying situations. To investigate cellular signaling by protein kinases, we developed monoclonal antibodies, designated as Multi-PK antibodies, that can recognize multiple protein kinases in various biological species. These Multi-PK antibodies can be used to profile the kinases expressed in cells and tissues, identify the kinases of special interest, and analyze protein kinase expression and phosphorylation state. Here we introduce some applications of Multi-PK antibodies to identify and characterize the protein kinases involved in epigenetics, glucotoxicity in type 2 diabetes, and pathogenesis of ulcerative colitis. In this review, we focus on the recently developed technologies for kinomics studies using the powerful analytical tools of Multi-PK antibodies.

Keywords: 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; CDKL5, cyclin-dependent kinase-like 5; CNBr, cyanogen bromide; CaMK, Ca2+/calmodulin-dependent protein kinase; DCLK, double-cortin like protein kinase; Dnmt1, DNA methyltransferase 1; FAK, focal adhesion kinase; IEF, isoelectric focusing; IPG, immobilized pH gradient; Kinomics; MAPK, mitogen-activated protein kinase; MeCP2, methylated-CpG-binding protein 2; Monoclonal antibody; Protein kinase; Protein phosphorylation; Proteomics.

Fig. 1

Amino acid sequences of the subdomain VIB and the peptides used as antigens for the generation of Multi-PK antibodies. (A) Alignment of subdomain VIB sequences in various protein kinases and the peptides used as antigens: 16PEN for M1C and M8C antibody production; 11RAAN for YK34 antibody production. (B) Amino acid sequences of subdomain VIB in Ser/Thr protein kinases in plant. The clones for Ser/Thr kinases were isolated from a cDNA library of L. japonicus by expression screening using the M1C/M8C antibodies.

Fig. 2

Schematic illustrations of the analytical methods for protein kinases using Multi-PK antibodies. (A) Schematic illustration of the 2D-PAGE analysis for profiling of protein kinases. The expression pattern of the protein kinases was analyzed by Western blotting with Multi-PK antibodies after separation by 2D-PAGE using a MicroRotofor in the first dimension. (B) Outline of the 2D-PAGE analysis to detect subdomain VIB-containing fragments. The procedure consists of the first SDS-PAGE followed by in-gel CNBr digestion, secondary SDS-PAGE, and Western blotting with Multi-PK antibodies. (C) Schematic representation of the phosphorylation state analysis. Western blotting analysis using Multi-PK antibodies was performed after separation by SDS-PAGE (left panel) and Phos-tag SDS-PAGE (right panel). PK: protein kinase; P-PK: phosphorylated protein kinase.

Fig. 3

Isolation and identification of Dnmt1-binding kinases. (A) Outline of the isolation of Dnmt1-binding proteins. A mouse brain extract was applied to a Dnmt1(1-290)-affinity column, and the unbound proteins were eluted out with an equilibration buffer. The Dnmt1-binding proteins were eluted with 0.3 M NaCl. (B) The protein kinases in each fraction were analyzed by Western blotting with the M8C Multi-PK antibody. The arrowhead indicates the migration position of CDKL5, which was identified by LC-MS/MS analysis.

Fig. 4

Identification of a protein kinase correlated with insulin secretion. (A) Effect of glucose concentration on basal insulin secretion in INS-1 cells. Secretion of insulin was assessed by sandwich-type ELISA, and the data represent means ± SE (n = 3) of separate experiments. (B) The protein kinases (left panel) and CaMKIV (right panel) in INS-1 cells cultured under varying glucose concentrations were detected by Western blotting using the M8C Multi-PK antibody and an anti-CaMKIV antibody.