Abstract
Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine kinase that is activated following an increase in the intracellular Ca2+ concentration and activates multiple signaling cascades that control physiologically important neuronal processes. CaMKK2 has been implicated in schizophrenia, bipolar disease, neurodegeneration, and cancer. Using isoelectric focusing (IEF) and mass spectrometry-based proteomic analysis, it was found that knockdown (KD) of CaMKK2 in cultured adult primary dorsal root ganglion (DRG) neurons resulted in the reduction of transferrin (TF) phosphorylation at multiple functionally relevant residues which corresponded to loss of an acidic fraction (pH~3-4) of TF. In vitro studies using CRISPR/Cas9 based CaMKK2 knockout (KO) HEK293 and HepG2 cells lines validated previous findings and revealed that loss of CaMKK2 interfered with TF trafficking and turnover. TF is an iron transporter glycoprotein. Abnormal accumulation of iron and/or deregulated Ca2+ homeostasis leads to neurodegeneration in Alzheimer's disease (AD). Therefore, it was hypothesized that aberrant CaMKK2 in AD may lead to aberrant phosphorylated transferrin (P-TF: pH~3-4 fraction) which may serve as a hallmark biomarker for AD. A significant reduction of P-TF in the brain and serum of CaMKK2 KO mice and a triple-transgenic mouse model of AD (3xTg-AD) supported this hypothesis. In addition, analysis of early (< 65 years) and late-stage (>65 years) postmortem human AD cerebrospinal fluid (CSF) and serum samples revealed that aberrant P-TF (pH~3-4 fraction) profile was associated with both early and late-stage AD compared to age-matched controls. This indicates P-TF (pH~3-4 fraction) profile may be useful as a minimally invasive biomarker for AD. In addition, this study provides a link between aberrant CaMKK2 with TF trafficking and turnover which provides a novel insight into the neurodegeneration process.
Highlights
Calcium ions (Ca2+) are one of the most ubiquitous second messengers which play a crucial role in many signaling pathways, especially in neuronal tissues (Kawamoto et al, 2012)
In order to understand the role of calmodulin-dependent protein kinase kinase 2 (CaMKK2) in neurons, CaMKK2 was knocked down in cultured adult rat primary dorsal root ganglion (DRG) neurons (Figure 1A) and total cellular proteins were fractionated by isoelectric focusing (IEF)/SDS-PAGE to detect and compare differentially charged protein fractions between control and KD samples (Figure 1B)
This study reports that the relative amount of phosphorylated TF corresponding to an acidic fraction of pI/pH∼3-4 may serve as a potential biomarker for Alzheimer’s disease (AD)
Summary
Calcium ions (Ca2+) are one of the most ubiquitous second messengers which play a crucial role in many signaling pathways, especially in neuronal tissues (Kawamoto et al, 2012). Calmodulin (calcium-modulated protein: CaM) is an intracellular Ca2+ receptor which is able to bind four Ca2+ ions (Swulius and Waxham, 2008). The Ca2+/CaM dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine (Ser/Thr) kinase that becomes activated by Ca2+/CaM binding (Racioppi and Means, 2012). Protein profiling followed by mass spectrometric analysis revealed a significantly reduced acidic fraction (pI/pH∼3-4) of the transferrin (TF) containing multiple phosphorylated serine (Ser), threonine (Thr) and tyrosine (Tyr) residues in the CaMKK2 KD neurons compared to the control
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