Product Pathways - Lymphocyte Signaling
Pim-2 Kinase #7575
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant full length human Pim-2 kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human Pim-2 (Met1-Pro311) (GenBank Accession No. NM_010208) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-Pim-2 fusion protein was analyzed using SDS/PAGE followed by anti-Pim-2 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. Pim-2 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: R11-S6-peptide, 5 µg/50 µl, and 200ng/50 µl Recombinant Pim-2.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Pim-2 kinase activity: DELFIA® data generated using Phospho-Bad (Ser112) Antibody #9291to detect phosphorylation of substrate peptide (#1342) by Pim-2 kinase. In a 50 µl reaction, increasing amounts of Pim-2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-Pim-2 fusion protein is 64 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Pim-2 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Pim-2 activity using HTScan™ Pim-2 Kinase Assay Kit #7572 [Fig.3].
Background
Pim proteins (Pim-1, Pim-2 and Pim-3) are oncogene-encoded serine/threonine kinases (1). Pim-1, a serine/threonine kinase highly expressed in hematopoietic cells, plays a critical role in the transduction of mitogenic signals and is rapidly induced by a variety of growth factors and cytokines (1-4). Pim-1 cooperates with c-Myc in lymphoid cell transformation and protects cells from growth factor withdrawal and genotoxic stress-induced apoptosis (5,6). Pim-1 also enhances the transcriptional activity of c-Myb through direct phosphorylation within the c-Myb DNA binding domain as well as phosphorylation of the transcriptional coactivator p100 (7,8). Hypermutations of the Pim-1 gene are found in B-cell diffuse large cell lymphomas (9). Phosphorylation of Pim-1 at Tyr218 by Etk occurs following IL-6 stimulation and is correlated with an increase in Pim-1 activity (10). Various Pim substrates have been identified; Bad is phosphorylated by both Pim-1 and Pim-2 at Ser112 and this phosphorylation reverses Bad-induced cell apoptosis (11,12).
Pim-2 is highly homologous to Pim-1 with similar oncogenic functions (13,14). Three isoforms of Pim-2 can be generated for alternative start sites which run at 34, 38, and 40 kDa (13). Pim-2 leads to resistance to a variety of apoptotic stimuli and its expression is negatively regulated by growth factor withdrawal (15,16). Increaesed levels of Pim-2 has also been observed in certain cancers (17,18).
- Mikkers, H. et al. (2004) Mol Cell Biol 24, 6104-15.
- Selten, G. et al. (1986) Cell 46, 603-11.
- Meeker, T.C. et al. (1987) J Cell Biochem 35, 105-12.
- Dautry, F. et al. (1988) J Biol Chem 263, 17615-20.
- Moroy, T. et al. (1993) Proc Natl Acad Sci USA 90, 10734-8.
- Lilly, M. and Kraft, A. (1997) Cancer Res 57, 5348-55.
- Leverson, J.D. et al. (1998) Mol Cell 2, 417-25.
- Winn, L.M. et al. (2003) Cell Cycle 2, 258-62.
- Pasqualucci, L. et al. (2001) Nature 412, 341-6.
- Kim, O. et al. (2004) Oncogene 23, 1838-44.
- Aho, T.L. et al. (2004) FEBS Lett 571, 43-9.
- Yan, B. et al. (2003) J Biol Chem 278, 45358-67.
- van der Lugt, N.M. et al. (1995) EMBO J 14, 2536-44.
- Breuer, M.L. et al. (1989) EMBO J 8, 743-8.
- Fox, C.J. et al. (2003) Genes Dev. 17, 1841-1854.
- White, E. (2003) Genes Dev. 17, 1813-1816.
- Cohen, A.M. et al. (2004) Leuk. Lymphoma 45, 951-955.
- Dai, H. et al. (2005) Prostate 65, 276-286.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
