Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 630 transducers. This array of signaling events leads to increased proliferation of connective tissue during development and in wound healing. Autophosphorylation also occurs on the insulin receptor to stabilize the active state and to generate a smaller number of binding sites, as illustrated in FIGURE 14.36. A key event is the Tyr phosphorylation of insulin receptor substrate (IRS) proteins, notably IRS1, on as many as a dozen sites. IRS1 takes over interactions with several signaling effectors that, in the case of PDGF, bind directly to the receptor. Among these targets is PI 3-kinase which leads to activation of Akt-2 and several essential metabolic actions of insulin (see 14.16 Lipids and lipid-derived compounds are signaling molecules). IRS proteins are also phosphorylated by serine/threonine protein kinases to modulate their signaling capability. Tyr phosphorylation often enhances the enzymatic activity of the associated proteins. Other proteins gain enhanced function primarily as a consequence of greater proximity to targets achieved by binding through their SH2 domains to phosphotyrosine sites either on the receptors or IRS adaptors. The precise actions of the many tyrosine kinase receptors are determined by the overlapping sets of signal transducers with which they interact, as well as by detailed differences in amounts of signal transducers, adaptor accessory proteins and receptor expression patterns (see Figure 14.43). Insulin signaling through IRS1 Insulin CYTOPLASM Insulin receptor IRS1 PIP 2 p85 p110 PI 3-kinase PIP3 Akt signaling FIGURE 14.36 Insulin binding to its receptor causes activation of the receptor tyrosine protein kinase and autophosphorylation. The receptor kinase also phosphorylates IRS1, a large adaptor with many potential phosphorylation sites. IRS1 is an essential intermediate in insulin action. PI 3-kinase binds to IRS1 via the SH2 domain within its p85 subunit. Akt and PDK1 bind to PIP 3 produced by activated PI 3-kinase so that PDK1 can phosphorylate and activate Akt (see Figure 14.18). 14.31 Src family protein kinases cooperate with receptor protein tyrosine kinases Key concepts • Src is activated by release of intrasteric inhibition. • Activation of Src involves liberation of modular binding domains for activation-dependent interactions. • Src often associates with receptors, including receptor tyrosine kinases. The first protein tyrosine kinase to be discovered was Src, which was identified as the transforming entity in the Rous sarcoma virus. Src is the prototype of a number of related enzymes, the Src family kinases. It participates in signaling pathways regulated by numerous cell surface receptors, including those that lack their own kinase domain (see in 14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane). Src is bound to the plasma membrane via an N-terminal myristoyl group. In the inactive state, Src is phosphorylated on Tyr527, C- terminal to its catalytic domain, by CSK (C-terminal Src kinase). The structure and regulation of Src is depicted in FIGURE 14.37. Phosphorylation of Tyr527 causes it to bind to its own SH2 domain. The SH2 and SH3 domains suppress the kinase activity through interactions on the surface of the protein. The SH3 domain binds to an SH3 binding site distant from the active site. Activation of Src by dephosphorylation of Tyr527 causes its SH2 to dissociate; this causes a conformational change in the SH3 domain to dissociate it from the binding site. Viral isolates of Src are often truncated prior to Tyr527, which increases their activity. Conformational changes in the kinase domain resulting from dissociation of the SH3 promote Src autophosphorylation on Tyr416 in its activation loop and further increase protein kinase activity. An important consequence of the interaction of Src with its own SH2 and SH3 domains is that these domains cannot bind anything else when in the autoinhibited state; therefore, other interactions are promoted when the SH2 and SH3 domains are released from their associations with the Src kinase domain. The heterologous interactions of the SH2 and SH3 domains contribute to Src localization and signaling. 630 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 631 Structure and regulation of Src INACTIVE ACTIVE KINASE DOMAIN SH3 SH2 P P Tyr527 FIGURE 14.37 The structures of inactive and active Src are compared. The inactive protein is autoinhibited by binding to its own SH2 and SH3 domains. The SH2 domain binds to phosphorylated Tyr527. The SH3 domain binds to a noncanonical SH3-binding motif on the opposite side of the kinase domain active site. In contrast to the steric inhibition of PKA caused by its R subunit, inhibition of Src by its SH2 and SH3 domains is allosteric. In the active structure the SH2 and SH3 domains are not bound to the kinase domain and are available for heterologous interactions. Structures generated from Protein Data Base files 1FMK and 1Y57. 14.32 MAPKs are central to many signaling pathways Key concepts • MAPKs are activated by Tyr and Thr phosphorylation. • The requirement for two phosphorylations creates a signaling threshold. • The ERK1/2 MAPK pathway is usually regulated through Ras. Mitogen-activated protein kinases (MAPKs) are present in all eukaryotes. They are among the most common multifunctional protein kinases mediating cellular regulatory events in response to many ligands and other stimuli. MAPKs are activated by protein kinase cascades consisting of at least three protein kinases acting sequentially, as illustrated in FIGURE 14.38. Activation of a MAPK is catalyzed by a MAPK kinase (MAP2K), which is itself activated by phosphorylation by a MAPK kinase kinase (MAP3K). MAP3Ks are activated by a variety of mechanisms including phosphorylation by MAP4Ks, oligomerization, and binding to activators such as small G proteins. MAP2Ks are activated by phosphorylation on two Ser/Thr residues; MAP2Ks then activate MAPKs by dual phosphorylation on Tyr and Thr residues (Figure 14.30). Each MAP2K phosphorylates a limited set of MAPKs and few or no other substrates. The great specificity of MAP2Ks is one means of insulating MAPKs from activation by inappropriate signals. Both Tyr and Thr phosphorylations are required for maximum MAPK enzymatic activity. Studies on the MAPK ERK2 led to an understanding of the events induced by phosphorylation that are important for increased activity. Conformational changes include refolding of the activation loop to improve substrate positioning and realignment of catalytic residues; this is most obvious in the repositioning of helix C, which contains a Glu involved in phosphoryl transfer. Amplification occurs moving down the cascade from the MAP3K to the MAP2K step because the MAP2Ks are much more abundant than the MAP3Ks. The MAP2K to MAPK step may also amplify the signal if the MAPK is present in excess of the MAP2K. In addition, the phosphorylation of a MAPK by a MAP2K on a 14.32 MAPKs are central to many signaling pathways 631
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Principles of cell signaling - UT Southwestern

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