Insulin receptor autophosphorylation

McCallum CD, Epand RM. Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties. Biochemistry 1995 34 1815-1824. 

FIGURE 3.1 Early biochemical steps following insulin receptor activation. Following insulin binding and insulin receptor autophosphorylation and activation, insulin receptor substrates-1 and -2 (IRS-1 and -2) are phosphorylated allowing binding of p85 and pi 10 phosphatidylinositol kinases and the generation of inositol triphosphate (IP3). Source Redrawn from Thirone et al ... 

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. 

Concanavalin A is a plant lectin from the jack bean (Canavalia ensiformis) which binds with high affinity to mannose residues of glycoproteins. Concanavalin A is known to stimulate the tyrosine kinase activity of the INSR (3-subunit with consecutive activation of kinases downstream the insulin receptor (IRS, PI 3-kinase). It is believed that Concanavalin A stimulates the activation and autophosphorylation of the INSR kinase through aggregation of the receptor, although the precise mechanism of action is unclear. 

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. 

The insulin-like growth factor I receptor is closely related to the insulin receptor. The RTK activity of the IGF-I receptor is regulated by intermolecular autophosphorylation at three sites within the activation loop. The crystal structure of the trisphosphorylated form of IGF-I RTK domain with an ATP analog and a specific peptide substrate showed that autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Furthermore, the structure revealed how... 

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)...

It is not clear whether V(V) or V(IV) (or both) is the active insulin-mimetic redox state of vanadium. In the body, endogenous reducing agents such as glutathione and ascorbic acid may inhibit the oxidation of V(IV). The mechanism of action of insulin mimetics is unclear. Insulin receptors are membrane-spanning tyrosine-specific protein kinases activated by insulin on the extracellular side to catalyze intracellular protein tyrosine phosphorylation. Vanadates can act as phosphate analogs, and there is evidence for potent inhibition of phosphotyrosine phosphatases (526). Peroxovanadate complexes, for example, can induce autophosphorylation at tyrosine residues and inhibit the insulin-receptor-associated phosphotyrosine phosphatase, and these in turn activate insulin-receptor kinase. 

An important question arises about the effects of phospholipid composition and the function of membrane-bound enzymes. The phospholipid composition and cholesterol content in cell membranes of cultured cells can be modified, either by supplementing the medium with specific lipids or by incubation with different types of liposomes. Direct effects of phospholipid structure have been observed on the activity of the Ca2+-ATPase (due to changes in the phosphorylation and nucleotide binding domains) [37]. Evidence of a relationship between lipid structure and membrane functions also comes from studies with the insulin receptor [38]. Lipid alteration had no influence on insulin binding, but modified the kinetics of receptor autophosphorylation. 

Once autophosphorylation begins, a complex of other events ensues. An insulin receptor substrate (IRS-1) binds the receptor and is phosphorylated on tyrosine residues, allowing proteins with SH2 (src homology) domains to bind to the. phosphotyrosine residues on IRS-1 and become active. In this way, the receptor activates several enzyme cascades, which involve ... 

In comparison to the level of cellular serine or threonine phosphorylation, protein tyrosine phosphorylation occurs at quite low levels in normal cells but dramatically increases upon oncogenic transformation or stimulation. Since the first discovery in 1978 that the transforming protein from Rous sarcoma virus (pp60vsrc) exhibited intrinsic kinase activity/5 protein kinase activity has also been shown to be inherent to other growth factor receptors such as epidermal growth factor receptor and the insulin receptor,[6 91 and to involve autophosphorylation processes. The diverse biochemical activity exhibited by protein tyrosine phosphorylation has stimulated the development of chemical methods for the preparation of phosphorylated peptides for use as substrates in elucidating the biochemical and physiological activity of phosphorylated site(s). 

The first insight into the mechanism by which autophosphorylation controls the activity of the Tyr kinase was possible via the crystal structure of the Tyr kinase domain of the human insulin receptor (Hubbard et al., 1994). 

Fig. 8.7. Structure of the catalytic domain of the insulin receptor. The crystal structure of the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994) has a two-lobe structure that is very similar to the structure of the Ser/Thr-specific protein kinases. Structural elements of catalytic and regulatory importance are shown. The P loop mediates binding of the phosphate residue of ATP the catalytic loop contains a catalytically essential Asp and Asn residue, found in equivalent positions as conserved residues in many Ser/Thr-specific and Tyr-specific protein kinases. Access to the active center is blocked by a regulatory loop containing three Tyr residues (Tyrll58, Tyrll62 and Tyrll63). Tyrll62 undergoes autophosphorylation in the course of activation of the insulin receptor. MOLSKRIPT representation according to Kraulis, (1991).

The insulin receptor substrate IRS couples the insulin receptor to sequential effector molecules (review Ogawa et al., 1998). On binding of insulin to the insulin receptor, the tyrosine kinase activity of the receptor is stimulated. The IRS protein is phosphory-lated at several Tyr residues, which then serve as attachment points for sequential effector molecules as e.g. the Grb2-mSos complex, the P13-kinase and the protein tyrosine phosphatase SHP-2. The IRS protein also has a phosphotyrosine binding domain and a PH domain. Both modules are required for signal transduction in vivo. It is assumed that the PTB domain binds to autophosphorylation sites of the insulin receptor and that the PH domain is involved in membrane association of IRS. 

Insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues. 

FIGURE 12-7 Activation of the insulin-receptor Tyr kinase by autophosphorylation. (a) In the inactive form of the Tyr kinase domain (PDB ID 11RK), the activation loop (blue) sits in the active site, and none of the critical Tyr residues (black and red ball-and-stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between Tyr1162 and Asp"32, (b) When insulin binds to the a chains of insulin receptors, the Tyr kinase of each /3 subunit of the dimer phosphorylates three Tyr residues (Tyr"58, Tyr"62, and... 

Signal transduction The binding of insulin to the a-subunits of the insulin receptor induces conformational changes that are transduced to the 3-subunits. This promotes a rapid autophosphorylation of a specific tyrosine residue on each 3-subunit (see Figure 23.7). Autophosphorylation initiates a cascade of cellsignaling responses, including phosphorylation of a family of pro teins called insulin receptor substrate (IRS) proteins. At least four... 

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