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PI3 Kinases - Coggle Diagram

- - - - These results suggested that PIP3 was probably the crucial signalling molecule but that PI(3,4)P2 was probably also important
      - PIP3 is at 0.03microM but 2.0microM after stimulation, at the inner leaflet of PM this may result in a local conc of 2mM
    - - This PI3K was shown to phosphorylate PI, PI4P and PI(4,5)P2 in vitro
  - - - Shows there is a localisation of PIP3 signals, why this is the case is not currently understood though
    - - PIP3 causes signal transduction
        PI3P causes endosome maturation, autophagy and protein sorting to lysosomes
        PI(3,4)P2 causes clathrin-mediated endocytosis and potentially signal transduction
- - - - Avoids redundancy in pathways that results from multiple isoforms but there are serious toxicity issues
    - - mTOR has similar ATP binding pocket
        More effective but similar issues with toxicity
      - Due to its role in the negative feedback loop inhibition of mTORC can increase sensitivity to GFs
    - - p110a
        
        potential for personalised medicine given its prevalence
        
        Resistance achieved by loss of PTEN and increased role of p110b
      - p110b
        
        There is evidence it is a major drive of PTEN-null cancers in mice
      - p110d
        
        lymphoid-selective expression so can be used for haematological malignancies (improves specificity and especially good with B/T cell cancers and lymphoma)
        
        Idelalisib was based on the first high selective PI3Kd inhibitor described
        
        Has become apparent that its effects are not as simple as predicted
        
        It targets both malignant B cells and tumour microenvironment, resulting in ejection of the cancerous cells from their niche (Okkenhaug, 2016, Cancer discov)
        
        Theory has been developed that inhibition of p110d could inhibit Tregs and MDSCs to allow a high [CD8] at solid tumours
        p110d & p110g mediate an action through indirect stimulation of Th1 cells that activate/stimulate CD8 expression so they could be used together to treat solid tumours (Ali, 2014 & Henau, 2019, both Nature)
      - On-target toxic effects observed in p110a (insulin-resistance) and p110d (chronic inflammation - particular issue for idelalisib)
  - - - E545K and H1074R mutations are the most common and line up with where the kinase domain interacts with regulatory subunits (Burke, 2015, TIBS)
        
        These mutations are all on the nodes of the 3D structure and therefore they probably ordinarily help stabilise the structure
    - - Mutations in PIK3CA and PTEN are present in 40-50% of cancers, their roles are confirmed in transgenic mouse models (Okkenhaug, 2016, Cancer Discov)
        
        KO slows PIP3 degradation (there are other PIP3 phosphatases) but experiments have shown it to slow the rate of decline and increase peak [PIP3]
      - Mutations in one allele are seen in Cowden's disease - patients have a predisposition to cancer
      - ~70% of castration-resistant prostate cancers have PTEN mutations
        tissue-specific deletion in mice drives malignant transformation in prostate, thyroid and enometrium
- - - - Can be inhibited by wortmannin (covalent interaction) or LY294002 (non-covalent)
      - Pharma industry invested heavily in this, with selective inhibitors available for each type of PI3K p110 subunit, these drugs are now predominantly in oncological trials
    - - There are 20-30 different protein families that can bind PIP3, enabling this diversity of signalling target (Bilanges, 2019, Nat Rev Mol Cell Biol)
      - Slide 19 in lecture 1 gives highly comprehensive review of signalling pathways from PKB and how mTORC1 acts to provide feedback inhibition
    - - These findings allowed people to use PKB activation as a study of PI3K function (Stokoe, 1997, Science)
      - Alessi was able to identify the kinase (PDK-1) that mediated the phosphorylation of T308
        Sequencing showed that both PKB and PDK-1 had PH domains
        
        This functions by PDK-1 (has activity in the cytoplasm on PKC, PKA etc) binding PIP3 at the PM.
        PKB also binds PIP3 at the membrane, which derepresses it. PDK-1 then phosphorylates T308 due to close proximity and mTORC2 phosphorylates S473 to exert a finer level of control (only recently been shown though)
        Much of this has been shown in Lucic, PNAS, 2018
    - - Consist of heterodimers containing p110a/b/g/d which give the complex its name plus one regulatory subunit
        
        Class IA consists of PI3Ka, b & d, which all receive regulation from RTyrKs
        
        alpha and beta are ubiquitous, delta is haematopoetic
        Beta can be activated by GPCRs via coincidence activation
        
        Regulatory subunits are p85a/b, p55a/g, p50a
        
        SH3 and BH domains are the main source of differentiation in IA regulatory subunits but their function is unclear, although SH3 binds specific PolyPro sequences and BH may bind small GTPases.
        Each also has three SHR domains which bind P-Y in YXXM motifs
        
        SH2 domains bind RTyrKs through these YXXM motifs
        
        By mutating these SH2 domains it is possible to see which domains are important to the repression of each PI3K class (Burke, 2013, Adv Biol)
        This shows p110a is repressed by nSH2 and p110b&g are repressed by both n&cSH2
        
        Activation is the result of both derepression and increased local concentration by sequestering PI3K and its targets to the PM (Burke, 2018, Mol Cell)
        
        Ordinarily p85/55/50 SH2 domains make contacts with the kinase domain of p110
        
        p110b, d have extra C-lobe/c-SH2 domain interactions but weaker helical/n-SH2 interactions
        
        ABD of catalytic subunits binds regulatory subunit and RBD binds small GTPases (a = GTP-Ras, b = GTP-Rac/Cdc42 & bg subunit of G protein, d =~ GTP-Ras)
        
        Activation of alpha and delta occurs by Ras-GTP and P-Y independently binding PI3K to synergistically stabilise the active membrane-bound form
        
        Activation of beta occurs by similar action of P-Y and Gbg (alongside potentially GTP-Rac/Cdc42)
        
        Class IB is just PI3Kg, which receives regulation from GPCRs
        
        p101 and p84 are the regulatory subunits
        all class IB complexes are haematopoetic
        
        in vitro studies with recombinant proteins show that both heterodimers can be activated by Gbg and GTP-Ras but that the p101-dimer is more sensitive to Gbg than the p84-dimer
        
        Regulatory subunits do not repress basal activity but rather allow for signals to activate class IB PI3Ks (Vadas, 2013, PNAS & Suire, 2005, Cur Biol)
        
        D2O and mutagenesis studies have defined interfaces between Gbg and both p101 and p110g, also shows it does not directly bind p84
        
        Gbg and GTP-Ras activate synergistically (Burke, 2018 & Vadas, 2013 papers)
        
        KO of regulatory subunits gives ~same results in PI3K activity in response to fMLP but very different cellular responses in neutrophils (Deladeriere, 2015, Sci Signal)
        
        p101 seems to regulate cell movement and p84 NADPH oxidase
  - - - Arranged as:
        Complex I - Vps34, Vps15, Vps30/Atg 6 (beclin), Atg14
        Complex II - Vps34, Vps15, beclin, Vps38 (UVRag)
    - - Sorting nexins in protein sorting e.g. SNX1/4
      - WIPIs to induce autophagy e.g. WIPI1/2
      - Protein kinases involved in signalling e.g. SGK3
- - - - gamma KO protected SLE, RA, EAE and allergic asthma
      - delta KO protected RA, EAE and allergic asthma
      - Double KO was more resistant for RA than in individual KOs