BRES-D-13-00090R1
Expression of LC3 and Beclin 1 in the spinal dorsal horn following spinal nerve ligation-induced neuropathic pain
Prof. Dong Woon Kim
Enji Zhang; Min-Hee Yi; Youngkwon Ko; Hyun-Woo Kim; Je Hoon Seo; Young Ho Lee; Wonhyung Lee;
Apr 30, 2013
Dear Dr. Kim:
We are pleased to inform you that your manuscript has been accepted for publication in Brain Research.
2013년 4월 30일 화요일
2013년 4월 17일 수요일
2013년 4월 15일 월요일
PKB/Akt signaling (Cell 2007)
Figure 1. Upstream Activation of Akt by Growth FactorsAlso depicted is the complex relationship between Akt signaling and mTOR. Activated receptor tyrosine kinases (RTKs) activate class I phosphatidylinositol 3-kinase (PI3K) through direct binding or through tyrosine phosphorylation of scaffolding adaptors, such as IRS1, which then bind and activate PI3K. PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3), in a reaction that can be reversed by the PIP3 phosphatase PTEN. AKT and PDK1 bind to PIP3 at the plasma membrane, and PDK1 phosphorylates the activation loop of AKT at T308. RTK signaling also activates mTOR complex 2 (mTORC2) through a currently unknown mechanism, and mTORC2 phosphorylates Akt on the hydrophobic motif S473, which can be dephosphorylated by the S473 phosphatase PHLPP. Akt activates mTOR complex 1 (mTORC1) through multisite phosphorylation of TSC2 within the TSC1-TSC2 complex, and this blocks the ability of TSC2 to act as a GTPase-activating protein (GAP) for Rheb, thereby allowing Rheb-GTP to accumulate. Rheb-GTP activates mTORC1, which phosphorylates downstream targets such as 4E-BP1 and the hydrophobic motif on the S6 kinases (S6Ks; T389 on S6K1). PDK1 phosphorylates the activation loop on the S6Ks (T229 on S6K1) in a reaction independent of PDK1 binding to PIP3. Akt can also activate mTORC1 by phosphorylating PRAS40, thereby relieving the PRAS40-mediated inhibition of mTORC1. Once active, both mTORC1 and S6K can phosphorylate serine residues on IRS1, which targets IRS1 for degradation, and this serves as a negative feedback mechanism to attenuate PI3K-Akt signaling. See text for references to recent reviews detailing Akt regulation and mTOR signaling.
Figure 2. Cellular Functions of Ten Akt SubstratesAkt-mediated phosphorylation of these proteins leads to their activation (arrows) or inhibition (blocking arrows). Regulation of these substrates by Akt contributes to activation of the various cellular processes shown (i.e., survival, growth, proliferation, glucose uptake, metabolism, and angiogenesis). As illustrated by these ten targets, a high degree of functional versatility and overlap exists amongst Akt substrates. See text for detailed descriptions of substrates and functions.
PKB activator
Full activation of PKB requires phosphorylationon Thr308 and Ser473 by upstream kinases such as 3-phosphoinositide-dependent kinase-1(PDK1), Mammalian target of rapamycin (mTOR) complex2 and DNA-dependent protein kinase (Hemmings and Restuccia, 2012; Laplante and Sabatini, 2012a; Laplante and Sabatini, 2012b).
1. PDK1
Initially, PDK1 was identified by its ability to phosphorylateThr-308 on PKBα (Hemmings and Restuccia, 2012), which has been shown to play a crucial role in normal and pathophysiological conditions. It has been well accepted that PDK1 activityis regulated by reversible phosphorylation on Ser241 in the activation loop of PDK1 (Casamayor et al., 1999). However, PDK1 appeared to be further activated by tyrosine phosphorylation on Tyr9 and Ty373/3736 following insulin and pervanadate stimulation (Park et al., 2001). Interestingly, Tyr9 phosphorylation of PDK1 is required for Tyr373/376 phosphorylation which is occurred at the plasmamembrane. Therefore Tyr9 phosphorylation of PDK1 can be used as marker for PDK1 activation, since further activation of PDK1 is correlated Tyr9 phosphorylation of PDK1 (Park et al., 2001).
2. mTORC2
3. DNA-PK
PKB inhibitor
1. PTEN
2. CTMP
Activation of AKT is initiated by membrane translocation, which occurs after cell stimulation and PtdIns(3,4,5)P3 (PIP3) production. Localization of AKT to the plasma membrane is accomplished by an interaction between its pleckstrin-homology (PH) domain and PIP3. At the membrane, association with carboxy-terminal modulator protein (CTMP) prevents AKT from becoming phosphorylated and fully active. Phosphorylation of CTMP by an as yet unidentified kinase releases CTMP from AKT and allows AKT to be phosphorylated by PDK1 and PDK2 at Thr308 and Ser473, respectively. Phosphorylation at these two sites causes full activation of AKT. C2, C2 domain; CD, catalytic domain; p85 BD, p85-binding domain.
Target of PKB
1. activated target of PKB
1. eNOS
2. IKK : Phophorylation of IkB & activation of NFkB
3. Mdm2
4. mTOR
2. Inactivted target of PKB
1. Bad
2. Forkhead family
3. GSK3beta
4. Caspase-9
5. P27kip1 and p130Rb2
NFkB1 and NFkB2 are members of the Rel/NFkB family of transcription factors that also includes RelA, c-Rel, and RelB. Rel/NFkB members regulate the expression of genes that participate in immune, apoptotic and oncogenic processes.
NFkB is predominantly localized in the cytoplasm as a complex with inhibitory IkB proteins and is released and translocated to the nucleus after phosphorylation of IkB. Both NFkB1 (105 kDa) and NFkB2 (100 kDa) are synthesized as precursor molecules that are proteolytically cleaved to 50 and 52 kDa active subunits.
NFkB appears to have contradictory functions in apoptosis and cell survival.
1. NFkB mediates the survival response of many signals by inhibiting p53-dependent apoptosis and up-regulating anti-apoptotic members of the Bcl-2 family, and caspase inhibitors such as XIAP, and FLIP.
2. In contrast, NFkB is also activated by apoptotic stimuli involved in DNA damage and mediates upregulation of pro-apoptotic genes such as TRAIL R2/DR5, Fas, and Fas ligand.
Mechanism of NF-κB action.
In this figure, the NF-κB heterodimer between Rel (p65) and p50 proteins is used as an example. While in an inactivated state, NF-κB is located in the cytosol complexed with the inhibitory protein IκBα. Through the intermediacy of integral membrane receptors, a variety of extracellular signals can activate the enzyme IκB kinase (IKK). IKK, in turn, phosphorylates the IκBα protein, which results in ubiquitination, dissociation of IκBα from NF-κB, and eventual degradation of IκBα by the proteosome.
The activated NF-κB is then translocated into the nucleus where it binds to specific sequences of DNA called response elements (RE). The DNA/NF-κB complex then recruits other proteins such as coactivators and RNA polymerase, which transcribe downstream DNA into mRNA, which, in turn, is translated into protein, which results in a change of cell function.[1][2][3]
Target landmarks
: 1. IκBα expression (If stress, IκBα decrease, If recover IκBα increase)
2. p65 translocation (determined by nuclear fraction of WB, or IHC)
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