Product Pathways - Neuroscience
CaMKI-γ Kinase #7456
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Description
Purified recombinant human CaMKI-γ (C-terminal truncation) 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 CaMKI-γ (C-terminal trunction) (GenBank Accession No. NM_020439) 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-CaMKI-γ fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. CaMKI-γ 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, 3 mM MgCl2, 30 ng/μl calmodulin, 0.5 mM CaCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Autocamtide 2, 300 ng/μL and recombinant CaMKI-gamma: variable.
Quality Control
The theoretical molecular weight of the GST-CaMKI-gamma fusion protein is 79 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. CaMKI-gamma 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).
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- Wei, F. et al. (2002) Nat. Neurosci. 5, 573-579.
Application References
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