Kinase-substrate interactions

The next class of cosubstrates for kinases consists of proteins. Protein kinases are very important in the regulation of cellular processes. Investigations of both these mechanisms and the roles that the protein kinases play might be aided by the use of selective inhibitors of these kinases. One indication of just how little is known about the characteristics of protein kinase-substrate interactions is the fact that many of these enzymes are named by their activators rather than by their substrates. 

Parang K, Kohn JA, Saldanha SA, Cole PA. Development of photo-crosslinking reagents for protein kinase-substrate interactions. FEBS Lett 2002 520 156-160. 

Figure 4. Literature based kinase substrate interaction map of proteins identified in the phosphoproteomic dataset by Collins et al.

The extracellular domain of the HER-2 neu molecule is the therapeutic target for the specific IgGlx humanized chimeric monoclonal antibody Trastuzumab (Herceptin ), approved 1998 and found to be effective in malignancies with HER-2 overexpression. An important aspect of this specific immunotherapy includes the efficiency against non-proliferating disseminated tumor cells (dormant cells), which are usually in the GO phase of the cell cycle, where conventional chemotherapy is less effective. The HER-2 neu molecule is also the target for specific tyrosine kinase inhibitors such as Tykerb (lapatinib ditosylate), which block the kinase-substrate interaction and the extracellular tyrosine kinase receptors on tumor cells. 

Tyrosine phosphorylated IRS interacts with and activates PI 3-kinase [3]. Binding takes place via the SRC homology 2 (SH2) domain of the PI 3-kinase regulatory subunit. The resulting complex consisting of INSR, IRS, and PI 3-kinase facilitates interaction of the activated PI 3-kinase catalytic subunit with the phospholipid substrates in the plasma membrane. Generation of PI 3-phosphates in the plasma membrane reemits phospholipid dependent kinases (PDKl and PDK2) which subsequently phosphorylate and activate the serine/threonine kinase Akt (synonym protein... 

N-Myristoylation is achieved by the covalent attachment of the 14-carbon saturated myristic acid (C14 0) to the N-terminal glycine residue of various proteins with formation of an irreversible amide bond (Table l). 10 This process is cotranslational and is catalyzed by a monomeric enzyme called jV-myri s toy 11ransferase. 24 Several proteins of diverse families, including tyrosine kinases of the Src family, the alanine-rich C kinase substrate (MARKS), the HIV Nef phosphoprotein, and the a-subunit of heterotrimeric G protein, carry a myr-istoylated N-terminal glycine residue which in some cases is in close proximity to a site that can be S-acylated with a fatty acid. Functional studies of these proteins have shown an important structural role for the myristoyl chain not only in terms of enhanced membrane affinity of the proteins, but also of stabilization of their three-dimensional structure in the cytosolic form. Once exposed, the myristoyl chain promotes membrane association of the protein. 5 The myristoyl moiety however, is not sufficiently hydrophobic to anchor the protein to the membrane permanently, 25,26 and in vivo this interaction is further modulated by a variety of switches that operate through covalent or noncovalent modifications of the protein. 4,5,27 In MARKS, for example, multiple phosphorylation of a positively charged domain moves the protein back to the cytosolic compartment due to the mutated electrostatic properties of the protein, a so-called myristoyl-electrostatic switch. 28 ... 

McNamara RK, Lenox RH Distribution of protein kinase C substrates MARCKS and MRP in the postnatal developing rat brain. J Comp Neurol 397 337-356, 1998 McNamara RK, Stumpo DJ, Morel LM, et al Effects of reduced myristoylated alanine rich C-kinase substrate expression on spatial learning in mutant mice transgenic rescue and interaction with gene background. Proc Natl Acad Sci U S A95 14517-14522, 1998... 

R. Herbst, M. S. Shearman, A. Obermeier, J. Schlessinger, and A. Ullrich. Differennal effects of W mutations on pl45c-kit tyrosine kinase activity and substrate interaction. J Bid Chem, 267 (19), 13210-13216,... 

Polte TR, Hanks SK. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate pl30Cas. Proc. Natl. 

Wang, J., Gambhir, A., Hangyas-Mihalyne, G., Murray, D., Golebiewska, U., McLaughlin, S. Lateral sequestration of phosphatidylinositol 4,5-biphosphate by the basic effector domain of mystoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J. Biol. Chem. 2002, 277, 34401-12. 

This enzyme catalyzes a reaction that appears to be very homologous to that of pyruvate kinase. One similarity other than the proposed mechanism is that this enzyme also requires two cations to elicit the most active form of the enzyme. Although calculations of kinetic activation data indicate that both GDP and metal-GDP (and GTP and metal-GTP) can both serve as substrates for the enzyme, the function of GDPrree as substrate has not been demonstrated, and if so, the metal-GDP complex is the more active form (79). Unlike pyruvate kinase, this enzyme does not require a monovalent cation. Kinetic activation studies indicate that the enzyme-bound metal facilitates the interaction of the substrates phosphoenolpyruvate and nucleotide with the enzyme, but not the substrate CO2. High-resolution NMR studies of both phosphoenolpyruvate and the nucleotide have demonstrated that in contrast to the classic case of pyruvate kinase, the substrates appear to bind in the second coordination sphere of the bound cation. These results indicate a much different role in the activation processes. The activation appears to be modulated by metal-bound water molecules. This interaction appears to be with both the phosphate of phosphoenolpyruvate (80) and the y-phosphate of GTP (79) [as indicated in Fig. 6 (80).] Thus, in the activation of phosphoenolpyruvate, the metal must increase the cationic character of the water molecule with which the phosphate of the substrate interacts. 

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