PI3K Signaling
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PI3K Signaling

The PI3K (Phosphatidylinositde-3-Kinase) family of enzymes regulate diverse biological functions in every cell type by generating lipid second messengers that ultimately results in the mediation of cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking and Glucose homeostasis. On the basis of structural similarities, the PI3K members are sub-divided into three classes; Class I, II and III. The Class I and II PI3Ks are found only in metazoa, whereas, Class III PI3Ks are also found in unicellular eukaryotes. The Class I PI3Ks are sub-divided into two groups-the Class IA and Class IB PI3Ks. Class IA PI3Ks are heterodimeric enzymes consisting of regulatory subunits (p85-Alpha, p85-Beta or p55-Gamma) and a catalytic subunit (p110-Alpha, p110-Beta or p110-Delta). There is only one catalytic subunit and one regulatory subunit for Class IB PI3K, which are known as p110-Gamma and p101, respectively. Each of the catalytic subunits associates with all of the regulatory subunits. The function of Class I PI3Ks is to catalyze the phosphorylation of the 3 hydroxyl subunit of Phosphoinositides. In vitro, all Class I PI3Ks are capable of phosphorylating PtdIns/PI (Phosphatidylinositol) to PtdIns(3)P, PtdIns(4)P/PIP (Phosphatidylinositol-4-phosphate) to PtdIns(3,4)P2 and PtdIns (4,5)P2/PIP2 (Phosphatidylinositol-4,5-bisphosphate) to PtdIns(3,4,5)P3/PIP3 (Phosphatidylinositol 3,4,5-trisphosphate), with PIP2 as the main substrate in vivo (Ref.1 & 2). The acronyms PIP2 and PIP3 are used to describe the substrate and product of Class I PI3Ks, respectively. The Class II PI3Ks are termed as PIK3C2Alpha (Phosphoinositide-3-Kinase-Class-2-Alpha Polypeptide), PIK3C2Beta and PIK3C2Gamma. Whereas, Class I PI3Ks reside mainly in the cytoplasm until recruited to active signaling complexes, the Class II PI3Ks are largely constitutively associated with membrane structures, including plasma membrane, intracellular membranes and somewhat surprisingly with nuclei. In vitro, Class II PI3Ks phosphorylate PtdIns and PIP, but, unlike Class I PI3Ks, not PIP2. Class II PI3Ks is not associated with a regulatory subunit, but they possess extended N- and C-termini relative to the Class I PI3Ks, which may serve this function. The Class III PI3K, PIK3C3 (Phosphoinositide-3-Kinase-Class-3) preferentially phosphorylates PtdIns to yield PtdIns(3)P, which recruits a distinct group of effector proteins with so-called FYVE or Phox (PX) domains. The catalytic subunit is complexed with a serine-threonine protein kinase, PIK3R4 (Phosphoinositide-3-Kinase-Regulatory Subunit Polypeptide-4)/p150, also known as the regulatory subunit. The major cellular function of the Class III PI3Ks is in intracellular trafficking. Class III along with Class II PI3Ks regulate various aspects of vesicle trafficking, and as such, there is an obvious potential for their involvement in activities such as antigen processing and cytotoxic responses that involve the directed sub-cellular transport of intracellular vesicles and their cargo (Ref.3).

The functional role of PI3K is controlled by several upstream factors. The upstream regulators like the Itgs (Integrins) do not possess a kinase domain or enzymatic activity but rely on specific ECM (Extracellular Matrix) ligands or proteins like (various Collagen, Elastin, Fibrillin, Fibronectin, Laminin and Proteoglycans), which interact with major cytoskeletal modulators like Talin, Vinculin and Pxn (Paxillin) in Integrin cytoplasmic tails to oligomerize and phosphorylate FAK, Src and CAS for recruitment of other SH2-containing protein, including the PI3K. Like Integrins, VEC and ECAD multi-component complexes recruit PI3K upon interaction with ECM proteins. In a similar manner Growth Factors and Growth Factor Receptors stimulate Ras by recruiting SOS, GRB2 and SHC to the membrane in order to activate PI3K or phosphorylate Src to recruit PI3K. Growth Factors along with other extracellular ligands like Hormones, Neurotrophic Factors and LTA (Lipoteichoic Acid) recruit SOS, GRB2, SHC and GAB1 to the membrane via RTKs for effective activation of FAK, Fyn, JAKs and IRS that in turn activate PI3K and Ras. Neurotrophic Factors and Cytokines via Cytokine Receptors also phosphorylate JAKs, IRS and Fes for enhancing PI3K activation (Ref.4). Further Cytokines along with ligands of TNF (Tumor Necrosis Factor) family and Growth Factors activate TNFRs (TNF Receptors) that contain one or more TIM (TRAF Interacting Motifs) in their cytoplasmic tails. This leads to recruitment of TRAF (TNF Receptor Associated Factor) family members (TRAF1 through TRAF6), and activation of signal transduction pathways such as PI3K via Src. However, GPCR (G-Protein-Coupled Receptor) through G-Proteins directly activate PI3K and also modulate its catalytic activity through stimulation of Ras under the influence of many extracellular factors (Chemokines, Hormones, fMLP (N-formyl-Met-Leu-Phe), Thrombin, Adenosine, S-1P (Sphingosine-1-Phosphate), Bdk (Bradykinin), ADP, UDP, etc to name a few). Stress response system like the SM (Sphingomyelin) response upstream to PI3K signaling, link diverse environmental stresses (e.g. UV (Ultraviolet), Ionizing Radiation, etc) to cellular effector PI3K through generation of the second messenger Ceramide by hydrolysis of SM. UV stress also enhance direct PI3K activation (Ref.5).

The PI3K signaling plays a vital role in B- and T-cell development, maturation and function in response antigen invasion. In B-cells, the tyrosine kinases SYK, BLK and Fyn becomes activated within seconds of antigen-receptor triggering and phosphorylate the co-receptor CD19 and BCAP (B-Cell PI3K Adaptor), which provide binding sites for PI3Ks. CD19 is primarily responsible for BCR signal transduction. Activated SYK, BLK and BCAP recruit c-Abl and activate B-cell associated co-receptor complexes. Apart from CD19 the major co-receptors consists of CD21, CD22, CD40 and CD81. These recruit Lyn that modulate PI3K action via activation of c-Cbl. Similarly in T-cells, activation of TCR (T-Cell Receptor)/CD3 (CD3 Antigen) complex along with associated molecules like CD4, CD8, CD28 and CD152 recruit ZAP70, Lck (Lymphocyte-Specific Protein-Tyrosine Kinase) and Fyn to activate TRIM and PI3K. T-cell activation also induces co-stimulatory molecule ICos which along with TRIM provide the docking sites for the SH2 (Src-Homology-2) domains of the regulatory subunit of PI3K. The Tyr-Xaa-Xaa-Met motifs in their cytoplasmic domains facilitate such interaction (Ref.6). The BCR and TCR signaling do activate PI3K through Ras and Vav. Vav activates Rac and CDC42 that contribute to PI3K activation, upstream to the signaling cascade. Besides these other important cell surface molecules like the TLRs (Toll-Like Receptors) viz., TLR1, TLR2, TLR4 and TLR6 and FcRs (Fc Receptors) (via recruitment of SYK and Lyn) up regulate PI3K (Ref.7).

The upstream effectors activate the regulatory subunits of PI3K to recruit Ras. Activation of regulatory subunits and subsequent binding to Ras brings the catalytic subunits to the membrane that mediate a plethora of biological effects through the action of its second messengers, the D3 phosphorylated phosphoinositides. All the classes of PI3Ks phosphorylate the D3 position of phosphoinositides. The Class I PI3Ks are in the main focus as they phosphorylate PIP2 to produce PIP3 at the inner leaflet of the plasma membrane, which are the major phosphoinositides in the mammalian systems. This process is reversed by PTEN, a lipid phosphatase that removes the D3 phosphate from PIP3. A similar inhibitory function is exhibited by SHIP upon FcR induction, which hydrolyzes PIP3 and hence interrupts the further downstream signaling by PI3K. Interaction of regulatory subunits of PI3K with adaptor protein Ruk or UV-stress activated p14(ARF) or with compounds like MSC (Se-Methylselenocysteine) and Wortmannin, also down-modulate PI3K signaling. PIP3 accumulation activates GEFs (Guanine Nucleotide Exchange Factors) connecting PI3K and Rac/CDC42/Rho activation. PIP3 and catalytic PI3K also activate Vav upstream to CDC42. This enables PI3K to control RhoGTPase Signaling in order to regulate Actin polymerization and cytoskeletal rearrangement. Other direct targets of PIP3 and catalytic PI3K include SOS1 and Rac. SOS1 exert a GEF activity toward both Ras and Rac. SOS1 and Vav activate Rac to stimulate MAP3Ks directly or through induction of PAK. PI3K, under the influence of BCR/c-Abl, activate MAPK cascade via aPKC (Atypical PKC)/Raf1/MAP2K route (Ref.2 & 8). MAP3K and MAP2K activation enhance the functioning kinases like JNKs, p38 and ERKs that regulate subsequent MAPK Pathways to control cell proliferation through induction of transcription factors (like NF-KappaB, E2Fs, Elk1, ATFs, Ets and CREB). These factors also regulate Opn (Osteopontin) gene expression that modulates cellular processes like immune homeostasis, ossification and myelination. Opn upon translocation to the exterior controls Integrin Pathway through ECM/Integrins interactions and also activates Cadherin to recruit the regulatory PI3Ks. PI3K thus divert Integrin signaling towards cell survival/growth, cell migration and tumor invasion through Opn gene regulation. PI3K mediates immune response through TLR signaling by activating substrate like IRAK4 (Ref.4).

PI3K also appears to play a role in the down-regulation of the CDK (Cyclin-Dependent Kinase) inhibitors p27(KIP1). Downregulation of p27(KIP1) is mediated, at least in part, via Akt1 phosphorylation and inactivation of the Forkhead family of transcription factors like FkhRL1. FkhRL1 transcriptionally activates p27(KIP1), therefore, PI3K/Akt activation downregulate p27(KIP1) transcription through FkhRL1 inactivation. PI3K mediated inhibition of Forkheads plays a vital role in cell survival as well as cell cycle entry, because FkhRL1 induce the expression of pro-apoptotic genes such as the Fas Ligand gene and controls Apoptosis (Ref.2). Another CDK inhibitor, p21(CIP1), is also a direct target of Akt1 phosphorylation. Akt1-mediated phosphorylation of p21(CIP1) results in its translocation from the nucleus, thus preventing its inhibition of nuclear CDKs. Despite their apparent inhibitory roles, p21(CIP1) and p27(KIP1) are also required for CDK-Cyclin complex assembly and activation. It appears that the up-regulation of G1-Cyclins and/or the sustained down regulation of p21(CIP1) and p27(KIP1) are required, at least in part, for the development of tumorigenic potential. Because PI3K appears to be involved in the regulation of these molecules, its activity likely plays an integral role in promoting malignancy (Ref.5).

PI3K/Akt1 promotes cell survival by phosphorylation of pro-apoptotic BAD along with FkhRL1. This prevents BAD to inhibit the action of anti-apoptotic proteins like BclXL and Bcl2. BCR and TCR signaling activate tyrosine kinases like Tec, BTK and ITK that up regulate BclXL activity. Bcl2 and Akt1 antagonize the regulation of Caspase Cascade through inhibition of Caspase9 and thereby prevent apoptosis. PI3K signals, in conjunction with ERK signaling, is required for transcriptional induction of CcnD1, which is essential for CDK-activation and thus for G1 progression. However PI3K/Akt1-mediated inhibition of GSK3, stabilizes the action of Ctnn-Beta, a transcriptional co-activator of CcnD1 and thereby controls cell cycle and Oncogenesis (Ref.5 & 9).  The GSK3 suppression is achieved by activated Akt1, which phosphorylates GSK3 at Serine residues 9 and 21. GYS, the rate-limiting enzyme in Glycogen Synthesis, is inactivated by progressive phosphorylation of up to nine residues by several kinases including GSK3. PI3K signaling pathway thus relieves the inhibitory constraints on GYS and allows Glycogen Synthesis to occur. Ras-mediated suppression of oncogene c-Myc-induced apoptosis through GSK3 also occurs through the activation of the PI3K/Akt1 pathway. The biological significance of the link between shear stress, PI3K/Akt1 signaling, and eNOS cannot be overstated. eNOS activation and release of NO (Nitric Oxide) control blood vessel vasodilatation and hence blood pressure regulation. NO also modulates other processes including platelet aggregation, platelet and leukocyte adhesion to the endothelium, vascular smooth muscle cell proliferation, and Angiogenesis (Ref.5).

PI3K signaling turns on other signaling pathways via PIP3/Akt/PDK-1 activation. The major ones include cell survival through Akt Signaling; regulation of cAMP Pathway through activation of PDE (Phosphodiesterase) to facilitate the conversion of cAMP (Cyclic Adenosine 3,5-monophosphate) to AMP in form of metabolic energy; regulates protein translation and cell growth/apoptosis via induction of mTOR Pathway and p70S6K Signaling, respectively. Akt phosphorylates mTOR resulting in p70S6K activation (Ref.2 & 10). Further PIP3 accumulation activates PDK-1 that efficiently phosphorylates Akt, and is sufficient to activate PKC in order to modulate p70S6K signaling, NF-KappaB translocation and enhancement of electrolyte transport via modulation of ion channels like VGKC (Voltage-Gated Potassium Channel), SCn (Sodium Channel) and ClCn (Chloride Channel) through SGK (Serum/Glucocorticoid-Regulated Kinase) activation. The phosphorylation mechanism turned on by PI3K is switched off by PTEN that converts PIP3 back to PIP2. However PKC, Tec, ITK and BTK activate PLC (Phospholipase-C) that utilizes PIP2 as a substrate for the generation of IP3 (Inositol-1,4,5-trisphosphate) and DAG (Diacylglycerol). DAG mediates the function of nPKCs (Novel PKC) and cPKCs (Conventional/Classical PKC) but not aPKCs, whereas IP3 enters the IP3 Pathway to promote Calcium mobilization (Ref.8 & 10).In addition to the above mentioned factors, understanding spatial and temporal aspects of PI3K functional redundancy and signal pathway cross talk is essential in determining PI3K signaling specificity. The PI3K-mediated cellular decision hence reflects the dynamics and sum of all the individual signaling systems. One of the major challenges in understanding the versatility of PI3K function in cell physiology is deciphering signaling specificity. Inhibition of PI3K signaling may offer a potential target for the development of anti-cancer therapies and other pharmacological inhibitors (Ref.1 & 2).