Protein kinase pseudosubstrate binding

If the inhibitory structural element is itself a part of the protein kinase, this is known as autoinhibition. The inhibitory structural elements often have the character of a pseudosubstrate. They possess a similarity to the proper substrate and can accumulate in the substrate binding site. Since the pseudosubstrate lacks a phosphate receptor, no phosphorylation takes place (review Kemp et al., 1994). 

Fig. 7.8. Functional domains of protein kinase C. The functional domains of protein kinase Ca and C8 are shown as a linear representation. The binding site for TPA lies in domain Cl. Domain C2 contains the Ca binding site. Protein kinase C8 lacks the C2 elements and thus regulation by Ca. According to Azzi et ah, (1992). Pseudosubstrate autoinhibitory sequence with pseudosubstrate character.

Figure 10.30. Binding of Pseudosubstrate to Protein Kinase A. The two arginine side chains of the pseudosubstrate form salt bridges with three glutamate carboxylates. Hydrophobic interactions are also important in the recognition of substrate. The isoleucine residue of the pseudosubstrate is in contact with a pair of leucine residues of the enzyme.

Figure 15.16. Protein Kinase C Activation. (A) The Cl domain of PKC, structurally organized around two bound zinc ions, binds diacylglycerol. (B) When the Cl domains bind to diacylglycerol in the membrane, the pseudosubstrate is pulled from the active site, permitting catalysis. Calcium-binding C2 domains help to localize PKC to the membrane.

Figure 10.17 Regulation of protein kinase A. The binding of four molecules of cAMP activates protein kinase A by dissociating the inhibited hotoenzyme (R2C2 into a regulatory subunit (R2) and two catalytically active subunits (C). Each R chain includes cAMIP-binding domains and a pseudosubstrate sequence.

Figure 10.18 Protein kinase A bound to an inhibitor. This space- filling model shows a complex of the catalytic subunit of protein kinase A with an inhibitor bearing a pseudosubstrate sequence. Notice that the inhibitor (yellow) binds in a deft between the domains of the enzyme. The bound ATP, shown in red, is in the active site adjacent to the inhibitor. [Drawn from lATRpdb.]... 

Cyclic AMP serves as an intracellular messenger in the transduction of many hormonal and sensory stimuli. Cyclic AMP switches on protein kinase A, a major multifunctional kinase, by binding to the regulatory subunit of the enzyme, thereby releasing the active catalytic subunits of PKA. In the absence of cAMP, the catalytic sites of PKA are occupied by pseudosubstrate sequences of the regulatory subunit. 

The members of the protein kinase C family are composed of a polypeptide chain with a molecular weight of 68 - 83 kDa. The N-terminal regulatory domains Cl and C2 and a C-terminal catalytic domain can be differentiated in the primary structure (Fig. 7.9b) of the conventional PKCs. In addition, a pseudosubstrate sequence with autoinhibitory function is located in the Cl region that binds to the substrate-binding... 

Previously, we looked at protein kinase A to illustrate binding and catalysis by the active site of an enzyme. This enzyme can exist as an inactive tetrameric protein composed of two catalytic subunits and two regulatory subunits. Each regulatory subunit contains a pseudosubstrate sequence that binds to the active site in a catalytic subunit. By blocking substrate binding, the regulatory subunit inhibits the activity of the catalytic subunit. 

Many protein kinases are maintained in an inhibited state by an autoinhibitory domain within the protein or as a separate inhibitory subunit (Kemp et al., 1994). Activation thus involves exposure of the catalytic site so that the protein substrate may bind and be phosphorylated. A number of autoinhibitory sequences have structural similarities to the consensus phosphorylation site in the respective protein kinase substrates. Thus, the enzyme s active site may be ster-ically blocked by the autoinhibitory domain via a pseudosubstrate mechanism, where residues in the catalytic core bind to residues in the autoinhibitory region or substrate. 

How does the binding of cAMP activate the kinase Each R chain contains the sequence Arg-Arg-Gly-A/a-Ile, which matches the consensus sequence for phosphorylation except for the presence of alanine in place of serine. In the R2C2 complex, this pseudosubstrate sequence of R occupies the catalytic site of C, thereby preventing the entry of protein substrates (see Figure 10.28). The binding of cAMP to the R chains allosterically moves the pseudosubstrate sequences out of the catalytic sites. The released C chains are then free to bind and phosphorylate substrate proteins. 

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