General Principles of Control

Allosteric Regulation

Allosteric regulation is a classic widespread mechanism of control of protein function; effectors bind to regulatory sites distinct from the active site, inducing conformational changes that profoundly influence the activity [7]. Allosteric effectors typically bear no structural resemblance to the substrate of their target protein. This form of regulation explains how end products of metabolic pathways could act at early steps of the pathway to exert feedback control. In protein kinases, allosteric control can be exerted by flanking sequences or separate subunits/proteins, such as, for example,

Figure 1 Ribbon diagram of the structure of the catalytic domain of PKA, with the various regulatory regions colored: activation loop, red; P-loop, blue; helix C, cyan. The bound ATP and peptide substrate (only seven residues are shown) are shown in stick representation in orange and green, respectively. The figure was generated using GRASP [32].

Figure 1 Ribbon diagram of the structure of the catalytic domain of PKA, with the various regulatory regions colored: activation loop, red; P-loop, blue; helix C, cyan. The bound ATP and peptide substrate (only seven residues are shown) are shown in stick representation in orange and green, respectively. The figure was generated using GRASP [32].

the N-terminal sequence in EphB2 receptor tyrosine kinase or cyclin in cyclin-dependent kinases (CDKs) influencing the orientation of the lobes and rotation of helix C.

Intrasteric Regulation

The term intrasteric regulation was introduced to describe autoregulation of protein kinases and phosphatases by internal sequences that resembled substrate phosphorylation sites ("pseudosubstrates") and acted directly at the active site [8]. It is now clear that this form of control is used widely and extends to diverse enzyme classes as well as receptors and protein targeting domains [6]. Intrasteric interactions typically suppress protein functions, and diverse mechanisms can be used for activation, including protein activators or ligands, phosphorylation, proteolysis, reduction of disulfide bonds, or combinations of these. The best examples of intrasterically regulated protein kinases are the large subfamily activated either by calcium-binding proteins (e.g., calmodulin-dependent kinases [CaMKs], titin, twitchin) or calcium directly in the case of the plant calcium-dependent protein kinases that contain a calcium-binding domain fused with the kinase domain. In twitchin kinase, a C-terminal autoregulatory sequence threads through the active site cleft between the two lobes of the protein kinase domain, making a plethora of contacts with the peptide substrate binding site and ATP-binding residues as well as residues essential for catalysis [9] that completely shut down kinase activity. The binding site of the activator S100A1 has been mapped to one portion of the autoinhibitory sequence [9,10]. A very similar mechanism of inhibition occurs in the related giant kinase titin; however, here a combination of phosphorylation and calmodulin (CaM) binding (to a site analogous to the S100A1 binding site in twitchin) is required to activate the enzyme [11]. The more distantly related CaMK-I is also activated by CaM binding to an autoregula-tory sequence, but the structure shows a modified mode of

Figure 2 A schematic diagram showing prototypic kinase active structure (PKA) and inactive structures (IRK and twitchin), highlighting the regulatory regions. The two lobes of the catalytic domain, activation loop (thick line), helix C (cylinder), the ATP binding site (ATP), and the autoreg-ulatory sequence (thick gray line) are shown.

Figure 2 A schematic diagram showing prototypic kinase active structure (PKA) and inactive structures (IRK and twitchin), highlighting the regulatory regions. The two lobes of the catalytic domain, activation loop (thick line), helix C (cylinder), the ATP binding site (ATP), and the autoreg-ulatory sequence (thick gray line) are shown.

inhibition where the autoregulatory sequence exits the active site before the P-loop and not over the activation loop, as is the case for twitchin kinase [12]. In p21-activated kinase (PAK), the autoregulatory sequence is located on a distinct "inhibitory switch domain" binding to the C-lobe of the kinase which both blocks the substrate binding site and causes various distortions to the kinase domain [13]. PAK is activated by the GTP form of the Rho family of G proteins. Other protein kinase families predicted to be intrasterically regulated include glycogen synthase kinase 3p, which is proposed to be autoinhibited. In this case, phosphorylation of Ser-9 causes the N-terminus to bind to the small lobe and direct the autoregulatory sequence into the active site [14]. The protein kinase C family and cGMP-dependent protein kinases also have autoregulatory sequences N-terminal to the catalytic domain; the activators are phospholipids and diacylglycerol, and cGMP, respectively [15].

Insulin receptor tyrosine kinase (IRK) has revealed a more subtle autoinhibitory mechanism with a tyrosine residue bound to the active site [5]. This tyrosine may be considered a transient pseudosubstrate and is ultimately autophosphorylated in response to insulin binding to the extracellular part of the receptor. Phosphorylation of this and two other tyrosine residues results in a rearrangement, allowing access to the active site [16]. A similar blocking of the active site by a tyrosine is also observed in the inactive structure of the MAP kinase ERK2 [17]. In this case, the tyrosine is one of the two residues phosphorylated by a distinct upstream kinase to yield the active enzyme [18].

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