Proteins kinases

Protein kinase C (PKC) is an non receptor serine/threonine kinase which serves important subcellular functions and has an important role in a spectrum of adaptive and maladaptive cardiac responses. PKC is involved in the regulation of cardiac contractility, 

---

Manenti S, Sorokine O, Van Dorsseiaer A and Taniguchi H 1992 Affinity purification and characterization of myristoyiated aianine-rich protein kinase C substrate (MARCKS) from bovine brain J. Biol. Chem. 267 22 310-15... 

We have previously calculated conformational free energy differences for a well-suited model system, the catalytic subunit of cAMP-dependent protein kinase (cAPK), which is the best characterized member of the protein kinase family. It has been crystallized in three different conformations and our main focus was on how ligand binding shifts the equilibrium among these ([Helms and McCammon 1997]). As an example using state-of-the-art computational techniques, we summarize the main conclusions of this study and discuss a variety of methods that may be used to extend this study into the dynamic regime of protein domain motion. 

Calculation of Conformational Free Energies for a Model of a Bilobal Enzyme Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to protein substrates and are regulatory elements of most known pathways of signal transduction. 

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

As a template for an intermediate conformation of protein kinase, the crystal structure of the binary complex of cAPK with adenosine (Ibkx.pdb in the Protein Data Bank) was used. As templates for open conformations... 

Fig. 1. Superposition of three crystal structures of cAMP-dependent protein kinase that show the protein in a closed conformation (straight line), in an intermediate conformation (dashed line), and in an open conformation (broken line). The structures were superimposed on the large lobe. In three locations, arrows identify corresponding amino acid positions in the small lobe.

Fig. 2. Conformational free energy of closed, intermediate and open protein kinase conformations. cAPK indicates the unbound form of cAMP-dependent protein kinase, cAPKiATP the binary complex of cAPK with ATP, cAPKiPKP the binary complex of cAPK with the peptide inhibitor PKI(5-24), and cAPK PKI ATP the ternary complex of cAPK with ATP and PKI(5-24). Shown are averaged values for the three crystal structures lATP.pdb, ICDKA.pdb, and ICDKB.pdb. All values have been normalized with respect to the free energy of the closed conformations.

A number of issues need to be addressed before this method will become a routine tool applicable to problems as the conformational equilibrium of protein kinase. E.g. the accuracy of the force field, especially the combination of Poisson-Boltzmann forces and molecular mechanics force field, remains to be assessed. The energy surface for the opening of the two kinase domains in Pig. 2 indicates that intramolecular noncovalent energies are overestimated compared to the interaction with solvent. 

As examples of applications, we present the overall accuracy of predicted ionization constants for about 50 groups in 4 proteins, changes in the average charge of bovine pancreatic trypsin inhibitor at pH 7 along a molecular dynamics trajectory, and finally, we discuss some preliminary results obtained for protein kinases and protein phosphatases. 

The procedure is computationally efficient. For example, for the catalytic subunit of the mammalian cAMP-dependent protein kinase and its inhibitor, with 370 residues and 131 titratable groups, an entire calculation requires 10 hours on an SGI 02 workstation with a 175 MHz MIPS RIOOOO processor. The bulk of the computer time is spent on the FDPB calculations. The speed of the procedure is important, because it makes it possible to collect results on many systems and with many different sets of parameters in a reasonable amount of time. Thus, improvements to the method can be made based on a broad sampling of systems. 

The last part of this account will be devoted to protein kinases and protein phosphatases and some recent results we have obtained for them. Protein kinases and phosphatases are signaling biomolecules that control the level of phosphorylation and dephosphorylation of tyrosine, serine or threonine residues in other proteins, and by this means regulate a variety of fundamental cellular processes including cell growth and proliferation, cell cycle and cytoskeletal integrity. 

Left side of Fig. 4 shows a ribbon model of the catalytic (C-) subunit of the mammalian cAMP-dependent protein kinase. This was the first protein kinase whose structure was determined [35]. Figure 4 includes also a ribbon model of the peptide substrate, and ATP (stick representation) with two manganese ions (CPK representation). All kinetic evidence is consistent with a preferred ordered mechanism of catalysis with ATP binding proceeding substrate binding. 

Fig. 4. Ribbon model of protein kinase with peptide substrate and Mn2ATP (left) and protein phosphatase (right)...

Taylor, S. S., Radzio-Andzelm, E. Cyclic AMP-dependent protein kinase. In Protein Kinases, Woodgett, J. R., editor, IRL Press, Oxford, 1994. 

Karlsson, R., Zheng, J., Zheng, N.-H., Taylor, S. S., Sowadski, J. M. Structure of the mamalian catalytic subunit of cAMP-dependent protein kinase and an inhibitor peptide displays an open conformation. Acta Cryst. D 49 (1993) 381-388. 

Steinberg, R. A. A kinase-negative mutant of s49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunitof cyclic amp-dependent protein kinase. Mol. Cell Biol. 11 (1991) 705-712. 

This method has been successfully applied to the substituted indole 2.6B, an analogue of the teleocidin type of protein kinase activators[ll]. 

Protein feedstock Protein hydrolyzates Protein kinase... 

In addition, vinpocetine selectively inhibits a specific calcium, calmodulin-dependent cycHc nucleotide phosphodiesterase (PDF) isozyme (16). As a result of this inhibition, cycHc guanosine 5 -monophosphate (GMP) levels increase. Relaxation of smooth muscle seems to be dependent on the activation of cychc GMP-dependent protein kinase (17), thus this property may account for the vasodilator activity of vinpocetine. A review of the pharmacology of vinpocetine is available (18). 

Vanadium. Vanadium is essential in rats and chicks (85,156). Estimated human intake is less than 4 mg/d. In animals, deficiency results in impaired growth, reproduction, and Hpid metaboHsm (157), and altered thyroid peroxidase activities (112). The levels of coen2yme A and coen2yme Q q in rats are reduced and monoamine oxidase activity is increased when rats are given excess vanadium (157). Vanadium may play a role in the regulation of (NaK)—ATPase, phosphoryl transferases, adenylate cyclase, and protein kinases (112). 

The ANP leceptoi exists in two forms, ANP and ANPg, both of which have been cloned. These membrane-bound guanylate cyclases have a single transmembrane domain, an intracellular protein kinase-like domain, and a catalytic cyclase domain, activation of which results in the accumulation of cychc guanosine monophosphate (cGMP). A third receptor subtype (ANP ) has been identified that does not have intrinsic guanylate cyclase activity and may play a role in the clearance of ANP. 

The spatial and steric requirements for high affinity binding to protein kinase C (PKC), a macromolecule that has not yet been crystallized, were determined. Protein kinase C plays a critical role in cellular signal transduction and is in part responsible for cell differentiation. PKC was identified as the macromolecular target for the potent tumor-promoting phorbol esters (25). The natural agonists for PKC are diacylglycerols (DAG) (26). The arrows denote possible sites of interaction. 

Excitation of smooth muscle via alpha-1 receptors (eg, in the utems, vascular smooth muscle) is accompanied by an increase in intraceUular-free calcium, possibly by stimulation of phosphoUpase C which accelerates the breakdown of polyphosphoinositides to form the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 releases intracellular calcium, and DAG, by activation of protein kinase C, may also contribute to signal transduction. In addition, it is also thought that alpha-1 adrenergic receptors may be coupled to another second messenger, a pertussis toxin-sensitive G-protein that mediates the translocation of extracellular calcium. 

S Wang, DW Zaharevitz, R Sharma, VE Marquez, NE Lewm, L Du, PM Blumberg, GWA Milne. The discovery of novel, stnicturally diverse protein kinase C agonists through computer 3D-database pharmacophore search. I Med Chem 37 4479-4489, 1994. 

Conformational changes in a protein kinase are important for cell cycle regulation... 

---