Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

Abstract

Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied.

Individual Case Analysis of Postmortem Interval Time on Brain Tissue Preservation

“…A mouse expressing mutant tau has been shown to exhibit PMI effects on tau-5 staining, however the species difference or presence of the P301L mutation may explain this apparent discrepancy [ 18 ]. Others have shown that there is a significant loss of tau phosphorylation within minutes in both human biopsy samples [ 10 ] and in mice [ 22 ], and that this loss is exacerbated by not chilling immediately on ice, as substantial dephosphorylation occurs within the short time required for perfusion. Based on these studies, we can only assume that even at our earliest PMI of 4 h we are detecting diminished levels of tau phosphorylation to begin with, and indeed our findings agree with Matsuo et al [ 10 ] that dephosphorylation slows after time likely due to the diminution of the activity of phosphatases including PP2A.…”

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

“…Several studies suggest impaired signaling of PKA‐CREB in AD brain (Yamamoto‐Sasaki et al ., 1999; Puzzo et al ., 2005; Liu et al ., 2006). CREB is rapidly dephosphorylated during the postmortem interval (Wang et al ., 2015), but we could not obtain reliable results on the level of CREB phosphorylation in AD brain. Moreover, we recently found that PKA‐CREB signaling regulates the expression of neuron‐specific glucose transporter III (GLUTIII) (Jin et al ., 2013).…”

Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

“…repeat domain). Examples are cleavage by calpain or caspases (Wang et al, 2007, Flores et al, 2018 or non-enzymatic cleavage in aging cells which causes the heterogeneous "smear" in SDS gels of AD Tau (Watanabe et al, 2004). In support of this, incipient neurodegeneration correlates with the appearance of soluble tau oligomers rather than Taucontaining tangles (Lasagna-Reeves et al, 2012, Kaniyappan et al, 2017.…”