Product Pathways - Ca / cAMP / Lipid Signaling
PKD3/PKCν Kinase #7694
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Description
Purified recombinant full length human PKD3/PKCnu (Met1-Pro890) 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 human PKD3/PKCnu (Met1-Pro890) (GenBank Accession No. NM_005813) 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-PKD3/PKCnu fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. PKD3/PKCnu kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 40 ng/μL BSA, 50 μM ATP, Substrate: CREBtide 400 ng/μL and recombinant PKD3/PKCnu: variable.
Quality Control
The theoretical molecular weight of the GST-PKD3/PKCnu fusion protein is 126 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PKD3/PKCnu kinase activity was determined using a radiometric assay [Fig.2].
Background
PKCnu, also known as PKD3, is a member of the protein kinase C (PKC) family of serine/threonine kinases that play critical roles in the regulation of cellular differentiation and proliferation. PKCnu is composed of 890 amino acid residues and has 77.3% similarity to human PKCmu (PKCmu) and 77. 4% similarity to mouse PKD (the mouse homolog of PKCmu) (1). The PKCnu mRNA is ubiquitously expressed in various tissues. PKCnu has two putative diacylglycerol binding C1 domains, suggesting that it may participate in a novel diacylglycerol-mediated signaling pathway (2). PKCnu is translocated to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. PKCnu is an important physiologic target of the B-cell receptor (BCR) and exhibits robust activation after BCR engagement (2). GPCR agonists induce a rapid activation of PKCnu by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKCnu. PKCnu is present both in the nucleus and cytoplasm and this distribution of PKCnu results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway (3). Cell stimulation with the GPCR agonist neurotensin induces a rapid and reversible plasma membrane translocation of PKCnu that is PKC-dependent.
- Hayashi, A. et al. (1999) Biochim. Biophys. Acta. 1450, 99-106.
- Matthews, S.A. et al. (2003) J. Biol. Chem. 278, 9086-91.
- Rey, O. et al. (2003) J. Biol. Chem. 278, 23773-85.
Application References
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
