Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn 2؉ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3␤, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly upshifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC 50 value of 1.6 M in the presence of 0.10 mM ATP. Molecular & Cellular Proteomics 6:356 -366, 2007.Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1, 2). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4). T...

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE. The phosphate‐affinity site is a polyacrylamide‐bound Phos‐tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c‐Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr‐394, Tyr‐197, or Tyr‐18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two‐component system. FixL is autophosphorylated at the His‐285 with ATP, and the phosphate group is transferred to the Asp‐54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well‐known isotype phosphorylated at the Asp‐54. The other is a novel isotype phosphorylated at the His‐84.

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn(2+)-Phos-tag SDS-PAGE.

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scF(V)) mAb by using the variable heavy(V(H)) and light (V(L))genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrP(c) from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of V(H) and V(L) genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.

SUMMARYWe developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn 2+ -Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn 2+ -Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.