3- Phosphoinositide-dependent protein

The 3-phosphoinositide-dependent protein kinase-1 (PDKl) is a 556-amino acid enzyme composed of three well-differentiated motifs an N-terminal domain, a constitutively activated serine/threonine kinase domain, and a Pleck-strin homology (PH) domain at its C-terminus [87-91]. The attractiveness of PDKl as a potential anticancer target is hnked to its ability to control the activity of a diverse set of AGC kinase members, in particular the three PKB isoforms [92], Full activation of PKB requires phosphorylation at two sites. 

Alessi, D.R., Deak, M., Casamayor, A., Caudwell, F.B., Morrice, N., Norman, D.G., Gaffney, P., Reese, C.B., MacDougall, C.N., Harbison, D., et al. (1997). 3-Phosphoinositide-dependent protein kinase-1 (PDK1) structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7, 776-789. 

D. R. Alessi et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B alpha. Curr. Bid. 7, 261-269, 1997. 

D. R. Alessi, S. R. James, C. P. Downes, A. B. Holmes, P. R. J., Gaffney, C. B., Reese and P. Cohen. Characterization of a 3-phosphoinositide-dependent protein kinase wliich phosphorylates and activates protein kinase Ba. Curr Biol, 7, 261-269, 1997. 

H. et al.. Mouse 3-phosphoinositide-dependent protein kinase-1 undergoes dimerization and franx-phosphorylation in the activation loop, J. Biol. Chem. 278, 42913 2919, 2003 Wu, S. and Kaufman, R.J., trans-auto-phosphorylation by the isolated kinase domain is not sufQcient for dimerization or activation of the dsRNA-activated protein kinase PKR, Biochemistry 43, 11027-11034, 2004. 

Arico, S., Pattingre, S., Bauvy, C., Gane, P., Barbat, A., Codogno, P., and Ogier-Denis, E. (2002) Celecoxib Induces Apoptosis by Inhibiting 3-Phosphoinositide-Dependent Protein Kinase-1 Activity in the Human Colon Cancer HT-29 Cell Line, J. Biol. Chem. 277,27613-27621. 

Lali, F. V., Hunt, A. E., Turner, S. J., and Foxwell, B. M. (2000). The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independendy ofp38 mitogen-activated protein kinase. J. Biol. Chem. 275, 7395-7402. 

Protein kinase B (Akt) is a serine/threonine, mitogen-regulated protein kinase involved in the protection of cells from apoptosis as well as the promotion of cell proliferation and diverse metabolic responses. It is activated upon binding of phospholipids and phosphorylation at residues Thr and Ser by upstream kinases such as phosphoinositide-dependent protein kinase 1 and 2 (M15). Activation of... 

Activation of Akt kinase proceeds in a multi-step process with regulation by PtdIns(3,4,5)P3 as the critical step. PtdIns(3,4,5)P3 binds to the PH domain of Akt kinase and thereby recruits the enzyme to the cell membrane. This step is thought to be a prerequisite for a subsequent phosphorylation of a Thr residue in the activation loop (see Chapter 7 on protein kinases) and an N-terminal Ser residue of Akt. The protein kinase responsible for this step is named phosphoinositide-dependent protein kinase 1 (PDK1, review Vanhaesebroeck and Alessi, 2000), and this also contains a PH domain with high affinity for PtdIns(3,4,5)P3. Membrane targeting and the double phosphorylation activate Akt, allowing the phosphorylation of downstream substrates like the Bad protein (see Section 15.7.1), pro-caspase 9, the transription factor CREB (see Section 1.4.5.2), glycogen synthase kinase (GSK), and 6-phosphofructo-2-kinase. 

F. 11.14. The insulin receptor-protein kinase B signaling pathway. Abbreviations Ins, insulin IRS, insulin receptor substrate PH domains, pleckstrin homology domains PDKl, phosphoinositide-dependent protein kinase 1 PKB, protein kinase B. The final phosphorylation step that activates PKB is shown in blue. 

As illustrated in Table I, many hormones act by stimulating membrane-bound phospholipases. The most commonly affected enzyme is a phospholipase C with specificity for phosphoinositides, i.e., a phosphoinositidase C (PIC) and, among these, the most relevant has specificity for phosphatidylinositol bisphosphate yielding inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG act as second messengers to mobilize Ca2+ from intracellular stores and activate the phospholipid- and Ca2+-dependent protein kinase, respectively (protein kinase C) (for reviews see Refs. 87-90). A typical Gp-mediated response of this type occurs in neutrophils exposed to the chemoattractant peptide fMLP [91]. fMLP binds to specific membrane receptors which recognize proteolyzed fragments of bacterial pro-... 

Fig. 2. Messengers mediating the initial and sustained phases of the All-induced cellular response. Initial phase All-elicited hydrolysis of PIP2 induces a transient rise in cytosolic calcium (via IP3), a transient activation of calcium-, calmodulin-dependent protein kinases, a transient increase in the phosphorylation of early-phase phosphoproteins (Pra-P), and a transient cellular response. Sustained response All-elicited hydrolysis of phosphoinositides generates a sustained increase in the diacylglycerol (DG) content of the plasma membrane. In conjunction with a sustained increase in plasma membrane calcium cycling, DG induces the sustained activation of protein kinase C (CK), the sustained increase in the phosphorylation of late-phase phosphoproteins (P -P) and the sustained cellular response.

PBKs phosphorylate inositol lipids at the 3 position of the inositol ring to generate the 3-phosphoinositide PI(3,4,5)P3, (PIP3). PBKs of class IB are directly activated by GP t, but others PBK also belong to pathways of receptors with or coupled to tyrosine kinase activity. PIP3 may recruit to tlie membrane various protein kinases, phosphoinositide-dependent kinase-1 (PDK-1), protein kinase B (PKB, or Akt), protein kinase (PKCQ, PLC t and cytosolic tyrosine kinases such as the Bruton s tyrosine kinase (Btk). 

Figure 14.20 Insulin signaling. The binding of insulin results in the cross-phosphorylation and activation of the insulin receptor. Phosphorylated sites on the receptor act as binding sites for [insulini receptor substrates such as IRS-1. The lipid kinase phosphoinositide 3-kinase binds to phosphorylated sites on IRS-1 through its regulatory domain, then converts PIPj into PIPv Binding to PiP activates PIP3-dependent protein kinase, which phosphorylates and activates kinases such as Aktl. Activated Aktl can then diffuse throughout the cell to continue the signal-transduction palhway.

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