Tyrosine Src kinase

Figure 13.26 Schematic diagram of the SH2 domain from the Src tyrosine kinase with bound peptide. The SH2 domain (blue) comprises a central p sheet surrounded by two a helices. Three positively charged residues (green) are involved in binding the phosphotyrosine moiety of the bound peptide (red). (Adapted from G. Waksman et al.. Cell 72 779-790, 1993.)...

Src tyrosine kinases comprise SH2 and SH3 domains in addition to a tyrosine kinase... 

The polypeptide chain of Src tyrosine kinase, and related family members, comprises an N-terminal "unique" region, which directs membrane association and other as yet unknown functions, followed by a SH3 domain, a SH2 domain, and the two lobes of the protein kinase. Members of this family can be phosphorylated at two important tyrosine residues—one in the "activation loop" of the kinase domain (Tyr 419 in c-Src), the other in a short... 

Figure 13.30 Ribbon diagram of the structure of Src tyrosine kinase. The structure is divided in three units starting from the N-terminus an SH3 domain (green), an SH2 domain (blue), and a tyrosine kinase (orange) that is divided into two domains and has the same fold as the cyclin dependent kinase described in Chapter 6 (see Figure 6.16a). The linker region (red) between SH2 and the kinase is bound to SH3 in a polyproline helical conformation. A tyrosine residue in the carboxy tail of the kinase is phosphorylated and bound to SH2 in its phosphotyrosine-binding site. A disordered part of the activation segment in the kinase is dashed. (Adapted from W. Xu et al.. Nature 385 595-602, 1997.)...

Figure 13.31 Space-filling diagram of Src tyrosine kinase in the same view as Figure 13.30. The SH2 domain makes only a few contacts with the rest of the molecule except for the tail region of the kinase. The SH3 domain contacts the N-domain of the kinase in addition to the linker region. There are extensive contacts between the N- and C-domains of the kinase. (Adapted from W. Xu et al., Nature 385 596-602, 1997.)...

Src tyrosine kinase contains both an SH2 and an SH3 domain linked to a tyrosine kinase unit with a structure similar to other protein kinases. The phosphorylated form of the kinase is inactivated by binding of a phosphoty-rosine in the C-terminal tail to its own SH2 domain. In addition the linker region between the SH2 domain and the kinase is bound in a polyproline II conformation to the SH3 domain. These interactions lock regions of the active site into a nonproductive conformation. Dephosphorylation or mutation of the C-terminal tyrosine abolishes this autoinactivation. 

Devary, Y., Gottlieb, R.A., Smeal, T., Karin, M. (1992). The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell 71, 1081-1091. 

Figure 2 Depiction of the active ( open ) and inactive ( closed ) conformations of Src kinase based on the analysis of x-ray structures of c-Src tyrosine kinase crystallized in its inactive state [7]. The stabilization of the inactive conformation is influenced by multiple events including intramolecular binding of the tyrosine-phosphorylated C-terminus tail to the SH2 domain as well as interactions between the SH3 domain and the SH2-kinase linker. CT, C-terminal NT, N-terminal. 

Many non-receptor tyrosine kinases have been identified as products of retrovirally encoded oncogenes. Non-receptor tyrosine kinases can be divided into two groups transmembrane and cytosolic families. The c-src tyrosine kinase is the prototype of the cytosolic tyrosine kinases. Regions within these non-receptor tyrosine kinases share homology with the Src kinase, known as Src homology 2 and 3 (SH2 and SH3) domains, and mediate protein-protein interactions between the receptor tyrosine kinases and the intracellular targets (reviewed in Cantley et al., 1991 Pawson and Gish, 1992 Mayer and Baltimore, 1993). 

Rakhit, S., Conway, A-M., Tate, R., Bower, T, Pyne, N.J. and Pyne, S., 1999, Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle role of endothelial differentiation gene-1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem. J. 338 643-649. 

Fig. 3 X-ray structure of Src tyrosine kinase complexed with ATP-mimetic inhibitors AP23464 and AP23451 in active conformations of the protein (adapted from Dalgarno et al. [61])...

Finally, a strategy to exploit protein engineering to mutate the ATP-binding pockets of protein kinases with the objective of enhancing selectivity for synthetic ATP analogs or inhibitors has been developed [66-68] using Src tyrosine kinase as a prototype model. In brief, mutation of a conserved amino acid in the ATP binding pocket was made to create a unique new site... 

Fig.5 Schematic model of Src tyrosine kinase complexed with ATP and peptide substrate...

The crystal structures of the SH2 domains of Src tyrosine kinase and Lck tyrosine kinase in complex with Tyr phosphorylated peptides have enabled important insight to be obtained into recognition of the phosphotyrosine residue and the neighboring amino acids in class lA of SH2 domains. The phosphate residue is boimd in a deep pocket of the SH2 domain, at the end of which an invariant Arg residue (Arg PB5) is located which contacts the negatively charged phosphate by a two-pronged interaction. It can be estimated that a phosphoserine or phosphothreonine residue would be too short to enter into a similar interaction with the Arg residue. 

Several cases are described (Cohen et ah, 1995) in which binding of an SH2-containing enzyme to an activated receptor tyrosine kinase leads to increased catalytic activity of the enzyme. Examples are the PI3-kinase and phospholipase Oy. The mechanism of activation is not clear. It is possible, however, that the basis is an allosteric mechanism, as is assumed for activation of Src tyrosine kinase. The Src kinase can be phosphoryla-... 

An example of regulation of enzyme activity via SH3 domains is the negative regulation of the activity of Src tyrosine kinase by SH3-mediated interactions (review Cohen et al 1995). 

Some representatives of the retroviruses cause tumors in animals such as mice or chickens. The discovery of oncogenes initiated from the src gene of Rous sarcoma virus, which could be identified as the tumor causing principle of this retrovirus. The src gene codes for the Src tyrosine kinase (see 8.3). The gene sections of retroviruses responsible for tumor formation were designated oncogenes. 

Fan G, Shumay E, Malbon CC, Wang H (2001) c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization. J Biol Chem 276(16) 13240-13247 Ferguson G, Watterson KR, Palmer TM (2000) Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein coupled receptor kinases. Mol Pharmacol 57(3) 546-552... 

Mimnaugh EG, Worland PJ, Whitesell L, et al. 1995. Possible role for serine/threonine phsphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and pp60v-src tyrosine kinase. J Biol Chem 270 28654-28659. 

Antibody can be directed against a specific PI 3-kinase subunit (e.g., p85 a/ 3, pi 10 a/p/S, etc.), or either surface receptors (PDGF-R, the T-cell costimulatory molecule CD28) and cellular proteins (e.g., Src tyrosine kinases), which may coassociate with PI 3-kinase(s). Alternatively, the assays may be performed on any peptide fragments (suitably immobilized on beads) that have been used to precipitate cellular proteins. 

It is fitting to introduce the phospholipase pathways here because they are controlled by small G proteins. Phospholipases C and D (PLC, PLD) are controlled by Rho/Arf and Cdc42 and Src tyrosine kinases participate in the control of PLD. 36 Phospholipases form potent second messengers, such as inositol trisphosphate (IP3) and diacylglycerol (DAG). In Fig. 4.6a the reactions catalysed by phospholipases C and D and the connections between phospholipases C and D are summarized, and in Fig. 4.6b the regulatory pathways that activate phospholipase D synergistically are summarized.37... 

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