Phosphorylation autophosphorylation

Protein phosphorylation (autophosphorylation and activation of other proteins)... 

Insulin Receptor. Figure 1 Structure and function of the insulin receptor. Binding of insulin to the a-subunits (yellow) leads to activation of the intracellular tyrosine kinase ((3-subunit) by autophosphorylation. The insulin receptor substrates (IRS) bind via a phospho-tyrosine binding domain to phosphorylated tyrosine residues in the juxtamembrane domain of the (3-subunit. The receptor tyrosine kinase then phosphorylates specific tyrosine motifs (YMxM) within the IRS. These tyrosine phosphorylated motifs serve as docking sites for some adaptor proteins with SRC homology 2 (SH2) domains like the regulatory subunit of PI 3-kinase. 

The current understanding on activation of Tec kinases fits into a two-step model. In the first step an intramolecular interaction between the SH3 domain and aproline-rich region in the TH domain is disrupted by binding ofthe PH domain to phosphoinositides, G protein subunits, or the FERM domain of Fak. These interactions lead to conformational changes of Tec and translocation to the cytoplasmic membrane where, in a second step, Src kinases phosphorylate a conserved tyrosine residue in the catalytic domain thereby increasing Tec kinase activity. Autophosphorylation of a tyrosine residue in the SH3 domain further prevents the inhibitory intramolecular interaction resulting in a robust Tec kinase activation. 

Like all immunoreceptor family members, FceRI lacks intrinsic tyrosine kinase activity. IgE and antigen-induced crosshnking of FceRI initiates a complex series of phosphate transfer events via the activation of non-receptor Src, Syk and Tec family protein tyrosine kinases (fig. 1). The Src family kinase Lyn, which associates with the FceRI p subunit in mast cells, transphosphorylates neighboring FceRI ITAMs after receptor aggregation [7, 26]. Once phosphorylated, the p chain ITAM binds to the SH2 domain of additional Lyn molecules, while the phosphorylated y chain ITAM recruits Syk to the receptor complex, where it is activated by both autophosphorylation and phosphorylation by Lyn [2, 7,15, 26]. 

All RTKs contain between one and three tyrosines in the kinase activation loop, which is composed of subdomains VII and VIII of the protein kinase catalytic core. Phosphorylation of these tyrosines has been shown to be critical for stimulation of catalytic activity and biological function for a number of RTKs, including insulin receptor, FGF receptor, VEGF receptor, PDGF receptor, Met (hepatocyte growth factor receptor), and TrkA (NGF receptor). A major exception is the EGF receptor, for which autophosphorylation of a conserved tyrosine in the activation loop does not seem to be involved in signaling. Substitution of tyrosine with phenylalanine has no effect on RTK activity or downstream signals. 

In principle, RTK autophosphorylation could occur in cis (within a receptor monomer) or in trans (between two receptors in a dimer). In the first case, ligand binding would cause a change in receptor conformation that would facilitate c/ s-autophosphorylation of tyrosine residues located within or outside the PTK domain. In the second case, no conformational change must occur upon dimerization. The simple proximity effect would provide sufficient opportunity for trans-phosphorylation of tyrosines in the cytoplasmic domain by a second RTK. 

FIGURE 8.14 Activation of nonreceptor PTKs. Lck is held in an inaccessible compact state through phosphorylation of residue Y505, which interacts with the intramolecular SH2 domain. Upon activation of CD45 (ligand unknown), the pY505 is removed and the molecule opens up. Autophosphorylation at theY-394 residue in the catalytic domain constitutes the activation of the PTK. 

Figure 11.2 Structure of the insulin receptor (a). Binding of insulin promotes autophosphorylation of the (3-subunits, where each (3-subunit phosphorylates the other (3-subunit. Phosphate groups are attached to three specific tyrosine residues (tyrosines 1158, 1162 and 1163), as indicated in (b). Activation of the (3-subunit s tyrosine kinase activity in turn results in the phosphorylation of various intracellular (protein) substrates which trigger the mitogen-activated protein kinase and/or the phosphoinositide (PI-3) kinase pathway responsible for inducing insulin s mitogenic and metabolic effects. The underlying molecular events occurring in these pathways are complex (e.g. refer to Combettes-Souverain, M. and Issad, T. 1998. Molecular basis of insulin action. Diabetes and Metabolism, 24, 477-489)...

All isoforms of PKC are predominantly localized to the cytosol and, upon activation, undergo translocation to either plasma or nuclear membranes. However, newly synthesized PKCs are localized to the plasmalemma and are in an open conformation in which the auto inhibitory pseudosubstrate sequence is removed from the substrate binding domain. The maturation of PKC isoforms is effected by phosphoinositide-dependentkinase-I (PDK-I), which phosphorylates a conserved threonine residue in the activation loop of the catalytic (C4) domain [24]. This in turn permits the autophosphorylation of C-terminus threonine and serine residues in PKC, a step which is a prerequisite for catalytic activity (see also Chs 22 and 23). The phosphorylated enzyme is then released into the cytosol, where it is maintained in an inactive conformation by the bound pseudosubstrate. It was originally thought that 3-phosphoinositides such as PI(3,4)P2 and PI(3,4,5)P3 could directly activate PKCs. However, it now seems more likely that these lipids serve to activate PDK-1 (a frequent contaminant of PKC preparations). 

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