Product Pathways - Neuroscience
CaMKK2 Kinase #7396
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Description
Purified recombinant full length human CaMKK2 (Met1-Leu541) 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 CaMKK2 (Met1-Leu541) (GenBank Accession No. NM_172226) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using GSH-agarose.
Gel Staining
Figure 1. The purity of the GST-CaMKK2 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. CaMKK2 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 2.5 mM MnCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Myelin basic protein (MBP), 400 ng/μL, and recombinant CaMKK2: variable.
Quality Control
The theoretical molecular weight of the GST-CaMKK2 fusion protein is 88 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. CaMKK2 kinase activity was determined using a radiometric assay [Fig.2].
Background
The Ca2+/calmodulin-dependent kinase (CaMK) family, which is activated in response to elevation of intracellular Ca2+, includes CaMKI, CaMKII, CaMKIV and CaMK-kinases (CaMKKs) (1,2). CaMKI is a downstream substrate of CaMKK and has 4 isoforms: CaMKI-α, CaMKI-β, CaMKI-γ and CaMKI-δ. CaMKI is present in most cell types and may be involved in cellular functions including transcription, cytoskeletal organization, axonal growth cone motility and long-term potentiation in neurons (3,4,5,6). CaMKII is also ubiquitously expressed in most cell types. While muscular CaMKII has been linked to activation of mitochondrial biogenesis in muscle hypertrophy response, neuronal CaMKII regulates important neuronal functions, including neurotransmitter synthesis, neurotransmitter release, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, learning and memory and gene expression (7). Like CaMKI, CaMKIV is also a substrate of CaMKKs and is primarily restricted to the nucleus of neurons. CaMKIV regulates gene transcription in neurons through phosphorylation of transcription factors such as CREB and is required for fear memory (8).
CaMK kinase (CaMKK) is an upstream kinase for CaMKI and CaMKIV and is mostly expressed in neurons in brain tissue. CaMKK/CaMK cascade plays an important role in regulating Ca2+ mediated cellular response (9).
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