By Thomas Angel

Components that contribute to the cell signaling process that stimulates cell growth, death and division are of medical importance when attempting to isolate that causal effects of cancers and cell death. There is a class of phosphate kinases that have been implicated in a cell signaling pathways that affect cellular death and longevity as well as many other processes that have medically important implications. Phosphoinositide-3 kinase (PI3K) is involved in a phosphorylation of Phosphatidylinositol (Ptdlns) on the third carbon of the inositol ring as shown in figure 1. The phosphorylation of Ptdlns to Ptdlns-3-P, Ptdlns-3,4-P and Ptdlns-3,4,5-P is part of many signal cascades with in a cell (Fig 2).

Phosphatidylinositol (Ptdlns) is a component of eukaryotic cell membranes. Derivatives of Ptdlns are called phosphoinositides collectively. The inositiol ring can be phosphorylated on any of the free hydroxyl carbons (Fruman, 1998). Ptdlns are involved in signal transduction and are the precursors to many secondary messenger molecules. Ptdlns are involved in regulation of many cellular activities, such as, cell proliferation, survival, vesicle trafficking, glucose transport, and cytoskeletal organization.

There have been three mammalian PI3K genes cloned and they share 42-58% amino acid sequence identity, these are designated p110a , p110g and p110d . Each of these proteins contains an N-terminal region that interacts with regulatory protein subunits, a domain that binds a small G protein Ras, a PIK domain and a C- terminal catalytic subunit. The regulatory unit is called p85 because the molecular weight of the first two isoforms purified and cloned, p85a and p85g was 85 kDa. These subunits do not contain lipid kinase activity but contain domains that are modular, which can be separated functionally and spatially from the rest of the protein. (Fruman, 1998)

The p85 subunit is thought to play a role in cell transformation and cancer. The protein-tyrosine kinase, pp60-v-scr, is believed to be a retroviral-encoded oncogene that interacts with the p85 subunit of PI3K and causes cell transformation. Ptdlns-3-P has been shown to coprecipitate with the pp60-c-scr complex of polyoma-infected cells. The kinase was separated from the pp60-v-scr complex at high salt concentrations and in detergent solutions. In mutation studies it was found that all pp60-v-scr that failed to associate with Ptdlns failed to transform 3T3 fibroblasts, indicating that PI3K is an active participant in oncogenisis (Cantley, 1991)

There are three forms of PI3K and they differ in composition of subunits. In general PI3K is heterodimeric, it is composed of a regulatory subunit and a catalytic subunit. The three classes of PI3K kinases are differentiated based on their substrate specificity. Class I PI3K contains a catalytic subunit of 110 to 120 kDa which phosphorylates Ptdlns, Ptdlns4,5- bisphosphate and Ptdlns-4-P. Class I PI3K interact with the SH-2/SH-3 domain containing p85 proteins. The p85 subunit SH2/SH3 domains allow this regulatory subunit to interact with phosphorylated tyrosine residues in cell receptors and other molecules within the cell and change the binding affinity of the catalytic subunit in some instances. Class II PI3K phosphorylate Ptdlns and Ptdlns-4-P but not Ptdlns-4,5-bisphosphate. Class III PI3K are specific, they only react with Ptdlns and are structurally and chemically similar to yeast Vps34p, which is involved with trafficking of proteins from golgi to a vacuole in yeast (Vanhaesebroeck, 1997).

The DNA sequence of Vps34 was compared with the cDNA encoding for the p110 subunit of PI3K from bovine brain and was found to have a similar sequence. To further establish that the gene product of Vps34 was p110, researchers assayed for PI3K activity in wild type yeast cell extracts. Yeast cells with Vps34 gene deletion were also assayed along side yeast cells that over produced Vps34. PI3K activity was not present in cells with the Vps34 deletion and was high in cells that over expressed the gene product. In wild type cells, PI3K activity was seen. PI3K activity associated with the Vsp34 gene product is associated with trafficking of lysozomal enzymes in yeast (Schu, 1993).

There are two distinct activities that PI3K participates in, phosphorylation of the regulatory subunit by the catalytic subunit and lipid phosphorylation. The major substrates in regulatory self-phosphorlylation are serine residues. The p110 subunit phosphorylates p85a at serine 608, which results in a down regulation of the lipid kinase activity of the p110a subunit. The second kinase activity is lipid phosphorylation. PI3K phosphorylates Ptdlns, which passes a signal along in a cell signal cascade.

PI3K is inhibited by wortmannin, a fungal metabolite, which is an inhibitor of the lipid and protein kinase activities of the heterodimeric enzyme. The inhibition is irreversible and occurs by reacting covalently with lysine-802. The lysine residue is present in the p110a catalytic subunit. Lysine is a residue that is required for catalytic activity and this residue is conserved in all PI3K’s. ATP and ATP analogs effectively compete with wortmannin. Wortmannin is very potent and has been used to demonstrate the role of PI3K in diverse signal transduction processes. Another inhibitor is Ly294002 it reversibly inhibits PI3K.

PI3K has been studied extensively over the past eleven years. The result of this work has illustrated involvement of PI3K in a variety of cell signal pathways. PI3K is believed to be a proto-oncogene and regulate cell longevity. The work in defining PI3K’s role in these activities has lead to the development of schematics that show how PI3K is thought to fit into signal transduction related to apoptosis and its effects on other proteins in the signal transduction pathway.

Ras protein is an important regulator of cell growth. A Ras protein binds GTP, the Ras-GTP complex is the metabolically active form. When Ras has GDP bound it is metabolically inactive. Mitogenic effectors bind a cell receptor on the plasma membrane of a cell and stimulate Ras to bind GTP, the active form, which then stimulates Raf-1. Raf-1 is a serine/theronine kinase, which activates mitogentic effector kinase (MEK). MEK activates mitogen-activated protein kinase (MAPK). MAPK is a serine/threonine kinase, this activates transcription factors and other cytoplasmic factors leading to mitogenisis.

In work done to define Protein Kinase B’s (c-Akt) place in the signal cascade associated with cell apoptosis mutants were generated that were Ras dominant-negative or Raf-1 dominant-negative. Expression of either of these two genotypes individually inhibited MAPK. The activity of MAPK was inhibited by wortmannin, which inhibits PI3Ks. MAPK activity was nearly abolished when mutants PI3K, which lack lipid kinase activity, were substituted in solution. G protein Ras has also been shown to be involved in MAPK activity and signal transduction. A negative mutant Ras (N17Ras) suppressed the PI3K stimulation of MAPK cascade (Lopez-Ilasaca, 1997). A side note Raf is said to be a very potent oncogene. Research attempting to elucidate the role that PI3K plays in signal transductioon and its relationship with Ras was done. PI3K was fond to coprecipitate with Ras, which suggests that PI3K could be acting either as an affector or as a regulator of Ras. Ras proteins were bound to agarose beads, the Ras protein was then loaded with GDP or GTP and incubated with a purified PI3K. The activity of the bound PI3K protein was measured. It was seen that beads carrying wild type Ras protein loaded with a non-hydrolyzable analogue of GTP had high PI3K activities as compared to beads loaded with GDP. These results suggest that PI3K activity and rate of activity is dependant on activated Ras binding PI3K. Ptdlns-3 is induced by many growth factors including active Ras, which acts as a direct regulator of PI3K. ( Rodriguez-Viciana, 1994).

In Caenorhabditis elegans PI3K has been implicated in control of cell senescence and dauer formation. C. elegans goes into a state of suspended development in response to environmental stressors, this is called dauer formation. Longevity and dauer formation (daf) is regulated by the gene daf-23. The life span of C. elegans was observed, organisms with age-1(mg44) chromosome from both maternal and zygotic genotype were found to live 2.6 times longer than the wild type organisms. A comparison of age-1 gene amino acid content was done with mouse p110a . Age-1 cDNA sequence predicts a protein that is most similar to the PI3K p110, catalytic subunit. There are three classes of PI3K p110 subunits, designated p110a , p110g and p110d , that generate a membrane localized signaling molecule, Ptdlns, which is thought to transduce signals from upstream effectors like Protein kinase B (PKB). p110a is activated by regulatory p85 or p55 subunits that bind to the phosphorylated tyrosines on receptors and non-receptors. Age-1 may couple to a p85 like tyrosine kinase adapter rather than a heteromeric G protein. Age-1 is most closely related to p110a with 22% similarity (Morris, 1996).

PI3K blocks apoptosis by regulating Protein kinase B downstream. Protein kinase B is the cellular homologue of v-Akt, which causes cell transformation. Research from several laboratories has indicated the presence of signaling pathways from PI3K to serine/threonine protein kinases. PKB may mediate some cellular responses including protection from apoptosis. PI3K is thought to down regulate cell apoptosis by effecting Protein Kinase B activity which is believed to be the cell signaler that inhibits apoptosis. PKB activity is dependent on serum growth factors, which activate PI3K (Franke, 1997). PKB is a serine/threonine kinase, which is stimulated by insulin, platelet derived growth factor (PDGF), epidermal growth factor (EFG) and basic fibroblast growth factor (bFGF). Rat-1 fibroblasts or insulin receptor over expressing NIH3t3 cells expressed epitope tagged PKB (HA-PKB). Increased HA-PKB kinase activity was observed after treatment with PDGF, EGF, insulin or bFGF in these cells. The cells were treated with wortmannin and very limited HA-PKB activity was seen as a result which suggests that PI3K is involved in the activation of PKB. PKB activation mechanism was examined to see if its activation was due to phosphorylation. Phosphoamino-acid analysis showed that phosphorylation was on a serine residue of PKB. This phosphorylation was essential for PKB activity, which was shown by treating the active PKB immune complexes with a phosphatase that abolished the ability of PKB to phosphorylate substrates, and stopped its signal transduction activity. PKB activation of p70-s6k, which is a growth factor, by PDGF is inhibited by wortmannin and a genetic mutant p85 that is inactive.(Boudewijn, 1995). Over expression of PKB prevents apoptosis of neuronal cells that are induced by survival factor withdrawal or inhibition of PI3K. Studies have also shown that over expression of PKB blocks UV induced apoptosis in rat-1 cells (Franke, 1997).

PI3K is believed to be a proto-oncogene. DNA that encodes for a homologue of the bovine p110 catalytic subunit of PI3K has been shown to be a component of Avian Sarcoma virus 16 (ASV-16). This virus induced hemangiosarcomas in chickens and transformed chicken embryo fibroblasts (CEF’s) in cell culture. ASV-16 was characterized and a non-viral DNA insertion was found indicating that ASV-16 activity could be due to a possible oncogene of cellular origin. The 5’ end of the non-viral sequence of DNA was connected to viral gag sequence and the 3’ end was recombined with viral env gene. In transformed cells a monoclonal antibody for the gag sequence was used to establish that the transformed cells were expressing the inserted cloned DNA. With the aid of computer modeling it was determined that the protein encoded on the non-viral gene sequence was homologous to the catalytic subunit of bovine PI3K thus the genetic sequence is called v-P3K. The AVS-16 non-viral sequence (v-P3K) was cloned and introduced into the expression vector RCAS, this construct was transfected into CEF’s. The result was infectious retroviral progeny. Transformed cells were tested for the presence of the v-P3K- RCAS combination, it was present. In transfected cells a protein of about 150 kDa was present, this corresponds to the possible non-viral sequence v-P3K-RCAS product. Cells transfected with RCAS-v-P3K produced hemangiosarcomas, thus the link between PI3K and a possible role as a proto-oncogene (Chang, W. H et al.).

Ptdlns-3-P is involved in trafficking of proteins within the cell. Ptdlns-3,4,5-P3 has been implicated in signal transduction that leads to adhesion by interacting with the SH2, PH and C2 domains of proteins, actin rearrangement, and cell growth and cell survival. Ptdlns-3,4-p2 has been implicated in cells growth and cell survival by effecting PKC, PDK and PKB (Toker and Cantley, 1997).

The importance of continued study of PI3K is due to the role that this enzyme plays in cancer and its role as a secondary messenger in signal transduction. Understanding the mechanisms and interplay of PI3K with its substrates and products may one day prove useful in developing treatments for disease related to cell growth and signal transduction. The products of PI3K phosphorylations have been implicated in signal transduction cascades that are associated with a great variety of cell activities.

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Copyright © 1999 Thomas Angel and Koni Stone