CTMP/PDK1/PKB signaling pathway
PKB positive regulator
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 negative regulator
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
NFkB Pathway
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)