4. Introduction
• PI3-Kinases (phosphoinositide 3-kinases, PI3Ks) are family of
enzymes capable of phosphorylating the 3'OH of the inositol
ring of phosphoinositides. (PtdIns).
• They are responsible for coordinating a diverse range of cell
functions including proliferation, cell survival, degranulation,
vesicular trafficking and cell migration, which in turn are
involved in cancer.
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5. Discovery
• Lewis Cantley & colleagues - phosphoinositide kinase
associated with the polyoma middle T protein.
• Cantley and colleagues demonstrated that in vivo the enzyme
prefers PtdIns(4,5)P2 as a substrate, producing the novel
phosphoinositide PtdIns(3,4,5)P3.
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6. Classes
• Classifications are based on primary structure, regulation,
and in vitro lipid substrate specificity.
• Divided into three different classes:
• Class I,Class II, and Class III
• Class I :
• Responsible for the production of Phosphatidylinositol 3-
phosphate(PI(3)P), Phosphatidylinositol (3,4)-bisphosphate
(PI(3,4)P2), and Phosphatidylinositol(3,4,5)-
triphosphate(PI(3,4,5P3)
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7. • The PI3K is activated by G protein-coupled receptors
and tyrosine kinase receptors.
• Class I PI3K are heterodimeric molecules composed of a
regulatory and a catalytic subunit.
• They are further divided between IA and IB subsets on
sequence similarity.
• Class IA PI3K is composed of a heterodimer between a
p110 catalytic subunit and a p85 regulatory subunit.
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8. • Five variants of the p85 regulatory subunit, designated
p85α, p55α, p50α, p85β, and p85γ
• There are also three variants of the p110 catalytic
subunit designated p110α, β, or δ catalytic subunit.
• The p85 subunits contain SH2 and SH3 domains
• Class II :
• Class II and III PI3K are differentiated from the Class I by
their structure and function.
• Class II comprises three catalytic isoforms (C2α, C2β, and
C2γ)
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9. • Unlike Classes I and III, no regulatory proteins.
• Class II catalyse the production of PI(3)P and
PI(3,4)P2 from PI.
• C2α and C2β are expressed through the body, however
expression of C2γ is limited to hepatocytes
• The distinct feature of Class II PI3Ks is the C-terminal
C2 domain.
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10. • Class III:
• Class III produces only PI(3)P from PI
• More similar to Class I in structure, as they exist as a
heterodimers of a catalytic (Vps34) and a regulatory
(Vps15/p150) subunits
• Class III seems to be primarily involved in the
trafficking of proteins and vesicles
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11. Mechanism
• An assorted group of signalling proteins, containing PX
domain, pleckstrin homology domains(PH
domains), FYVE domains and other phosphoinositide-binding
domains, are recruited to various cellular membranes.
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12. Function
• Cell growth, proliferation, differentiation, motility, survival
and intracellular trafficking.
• Many of these functions relate to the ability of class I PI 3-
kinases to activate protein kinase B (PKB, aka Akt) as in
the PI3K/AKT/mTOR pathway.
• The p110δ and p110γ isoforms regulate different aspects of
immune responses.
• PI 3-kinases are also a key component of the insulin
signaling pathway.
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13. • Cancer :
• The class IA PI 3-kinase p110α is mutated in many
cancers.
• Many of the mutations cause the kinase to be more
active.
• The PtdIns(3,4,5)P3 phosphatase PTEN that antagonises
PI 3-kinase signaling is absent from many tumours.
• PI 3-kinase activity contributes significantly to cellular
transformation and the development of cancer
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14. • Learning & Memory :
• PI3K has also been implicated in Long-term
potentiation (LTP).
• Whether it is required for the expression or the
induction of LTP is still debated.
• PI3K inhibitors suppressed the induction, but not the
expression, of LTP in mouse hippocampal CA1.
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15. PI 3-Kinases as Protein Kinases
• Many of the PI 3-kinases appear to have a serine/threonine
kinase activity in vitro; however, it is unclear whether this
has any role in vivo.
• In addition to the class I – class III PI 3-kinases there is a
group of more distantly related enzymes that are sometimes
referred to as class IV PI 3-kinases.
• The class IV PI 3-kinases family is composed of ataxia
telangiectasia mutated (ATM), ataxia telangiectasia and
Rad3 related (ATR), DNA-dependent protein kinase(DNA-
PK) and mammalian Target Of Rapamycin (mTOR).
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16. Inhibitors as Therapeutics
• Wortmannin
• LY294002
• As therapeutics :
• Too toxic
• P110δ isoform specific
inhibitors, IC486068andIC87114, ICOS Corporation.
• GDC-0941 is a highly selective inhibitor of p110α with
little activity against mTOR.
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18. Introduction
• Rho-kinase has been identified as one of the effectors of the
small GTP-binding protein Rho.
• Vascular smooth muscle cell (VSMC) contraction ,actin
cytoskeleton organization, cell adhesion and motility,
cytokinesis, and gene expressions.
• Rho-kinase upregulates various molecules that accelerate
inflammation/oxidative stress, thrombus formation, and
fibrosis, whereas it downregulates endothelial nitric oxide
synthase.
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19. • VSMC hypercontraction, stimulates VSMC proliferation
and migration, and enhances inflammatory cell motility.
• Pathogenesis of vasospasm, arteriosclerosis, ischemia/
reperfusion injury, hypertension, pulmonary hypertension,
stroke and heart failure, and to enhance central
sympathetic nerve activity.
• Rho-kinase is an important therapeutic target in
cardiovascular medicine.
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21. • Rho family - Rho (isoforms A to E, and G), Rac (isoforms 1 to
3), Cdc42, and TC10.
• Rho-kinase α3/ROKα4/ROCK2
• Rho kinase substrates-Myosin-binding subunit (MBS) of
myosin phosphatase (MLCPh), ERM (ezrin, radixin, moesin)
family, adducin, intermediate filament (vimentin), Na+-
H+ exchanger, and LIM-kinase.
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22. Eeperimental Tools for Rho-Kinase
• 3 major domains
• Catalytic domain
• Coiled coil domain
• Putative pleckstrin – homology domain
• Rho-kinase activity is enhanced by binding GTP-Rho.
• Fasudil and Y-27632 have been developed and they inhibit
Rho-kinase activity in a competitive manner with ATP.
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24. Studies at Molecular Level
• Rho-kinase plays an important role in mediating various cellular
functions, not only VSMC contraction but also actin cytoskeleton
organization,cell adhesion and motility,cytokinesis, and, gene
expression, all of which may be involved in the pathogenesis of
arteriosclerosis/atherosclerosis.
• Arteriosclerosis is a slowly progressing inflammatory process of
arterial wall that involves all 3 layers.
• NO antagonizes the vasoconstrictor effect of Rho-kinase through
activation of myosin phosphatase.
• Rho-kinase also upregulates tissue factor in the intima and is
involved in endothelial contraction that increases endothelial
permeability and hence enhances atherosclerosis.
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28. Clinical Studies
• Fasudil is the only clinically available Rho-kinase
inhibitor at present, several other Rho-kinase inhibitors
are currently undergoing investigation.
• Intravenous form of fasudil is used for the treatment of
cerebral vasospasm only in Japan,its oral form is
undergoing clinical trials for angina pectoris in Japan and
Northern America.
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