Regulation of Protein Kinase Activity by Phosphorylation

Many serine/threonine protein kinases are regulated by changes in their phosphorylation state. Most of these protein kinases require phosphorylation of a conserved threonine residue within the so-called activation loop of the catalytic domain, which is located in the proximity of the kinase active site cleft, and in the linear sequence found between the DFG and APE motifs (Fig. 2A). This phosphorylated residue then interacts with multiple residues within the catalytic domain to modify the active site of the enzyme to a conformation suitable for substrate binding and catalysis. This is the simplest form of regula tion by phosphorylation and can occur via autophos-phorylation in the case of the cAMP-dependent protein kinase, or via the activity of an upstream kinase, discussed later for the activation of p70s6k or protein kinase B (PKB) by phosphatidylinositol-dependent protein kinase 1 (PDK1).

Additional levels of regulation by phosphorylation are also commonly employed. In contrast, the cyclin-dependent protein kinases are phosphorylated at an inhibitory site (Tyr15) in the glycine-rich loop that must be dephosphorylated by specific phosphatases (Cdc25) to permit activation. Many other protein kinases are reversibly phosphorylated on serine, thre-onine, and tyrosine residues outside the catalytic domain and in a large number of cases these are regulatory events that modulate the activation process. Phosphorylation thus provides one mechanism for exquisite control of these enzymes.

The related protein kinases, p70s6k and PKB, provide examples of the extremely complex and intricate

Protein Kinase Phosphorylation

Fig. 10. Intramolecular contacts between the twitchin kinase autoregulatory sequence and the catalytic core. Four major types of contacts occur between the autoregulatory sequence and the catalytic core. Protein-substrate-like contacts are shown in lined boxes. Contacts to residues involved in ATP binding are shown in gray boxes. Contacts to key catalytic residues are shown in black boxes or black ovals for the active site Asp residues. Contacts to residues in the activation loop region are unboxed.

Fig. 10. Intramolecular contacts between the twitchin kinase autoregulatory sequence and the catalytic core. Four major types of contacts occur between the autoregulatory sequence and the catalytic core. Protein-substrate-like contacts are shown in lined boxes. Contacts to residues involved in ATP binding are shown in gray boxes. Contacts to key catalytic residues are shown in black boxes or black ovals for the active site Asp residues. Contacts to residues in the activation loop region are unboxed.

control of kinase activity by phosphorylation. p70s6k plays a major role in regulating mRNA translation by phosphorylating the ribosomal protein S6, while PKB plays more diverse roles, including the regulation of glycogen metabolism and cellular survival. Despite their distinct roles, these two enzymes share many common features. Each is activated in response to a wide range of mitogenic signals including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, thrombin, and nerve growth factor (NGF). The activation of each kinase is dependent on the activity of phosphatidylinositol 3'-kinase (PI-3 kinase). Importantly, the activation of both p70s6k and PKB requires phosphorylation on equivalent sites within the catalytic domain (Thr229 and Thr308 respectively) and the C-terminus (Thr389 and Ser473, respectively). Each is also phosphorylated on another equivalent site (Ser371 and Thr451, respectively)

although the significance of this phosphorylation in regulating the enzymes is unclear (see Fig. 11).

3.2.1. P70s6k Phosphorylation and Structure

The activation of the p70s6k by numerous stimuli is associated with its phosphorylation at multiple sites, and can be visualized by polyacrylamide gel electro-phoresis that generates multiple, slower migrating bands. Treatment of p70s6k with phosphatase removes the slower migrating bands. A total of 10 phosphory-lation sites has been identified within the kinase sequence (Fig. 11A). These can be divided into functional groups, and evidence is accumulating of interdependence between phosphorylation sites. Ser411, Ser418, Thr421, and Ser424 that are all followed by proline residues are clustered within a 14-residue sequence that is an autoinhibitory sequence apparently acting via its interaction with the N-terminal Module I (see

P70s6k Structure

Fig. 11. Regulation of p70s6k and PKB. (A) Homologous regulatory phosphorylation sites in p70s6k and PKB. A schematic representation of the domain structure of each kinase is shown with the p70s6k autoinhibitory sequence in black. The homologous sequences surrounding the essential regulatory phosphorylation sites are aligned. (B) p70s6k phosphorylation sites. A schematic representation of p70s6k showing the location of all the known phosphorylation sites. The conserved catalytic domain is shown in black and the autoinhibitory domain is white. The linker region between these two domains contains two of the essential regulatory phosphorylation sites, Ser371 and Thr389. The locations of the proposed regulatory modules are indicated.

Fig. 11. Regulation of p70s6k and PKB. (A) Homologous regulatory phosphorylation sites in p70s6k and PKB. A schematic representation of the domain structure of each kinase is shown with the p70s6k autoinhibitory sequence in black. The homologous sequences surrounding the essential regulatory phosphorylation sites are aligned. (B) p70s6k phosphorylation sites. A schematic representation of p70s6k showing the location of all the known phosphorylation sites. The conserved catalytic domain is shown in black and the autoinhibitory domain is white. The linker region between these two domains contains two of the essential regulatory phosphorylation sites, Ser371 and Thr389. The locations of the proposed regulatory modules are indicated.

Fig. 11B). Phosphorylation of Thr229, Thr389, and Ser404 was identified on the basis of the sensitivity of these sites to the drug rapamycin. These sites are flanked by large hydrophobic residues suggesting they are phosphorylated by a common upstream kinase. Phosphorylation of Thr389 and Thr229 is essential for enzyme activity (see below). Very recently, three additional sites were identified (Thr367, Ser371, and Thr447), also with adjacent C-terminal proline residues. Like Thr229 and Thr389, Ser371 is homologous to residues conserved in many members of the protein kinase C family as well as Akt.

Structure-function analysis with individual sites substituted with acidic residues to mimic phosphory-lation, or with alanine revealed that Thr389, Thr229, and

Ser371 are essential for enzyme activity, while the autoinhibitory sites played a modulatory role. The kinase has been subdivided into four functional domains: an N-terminal domain (module I), the catalytic domain containing Thr229 (module II), a linker region containing Ser371 and Thr389 (module III), and a C-terminal domain containing the "autoinhibitory sites" (module IV). It appears that in the quiescent state, modules I and IV interact to inhibit phosphory-lation of Thr389. Binding of a postulated regulator breaks this interaction, allowing phosphorylation of the inhibitory sites in module IV that in turn allows phosphorylation of Thr389 (by a postulated protein kinase) and subsequently the final activating phos-phorylation at Thr229 by PDK1.

3.2.2. PKB Phosphorylation and Structure

As noted earlier, PKB also lies downstream of PI-3 kinase and the role of the PI-3 kinase products, PI(3,4,5)Ps and PI(4,5)P2, is better defined than for p70s6k. PI(3,4,5)P3 and PI(4,5)P2 play a dual role in the activation of PKB. Phospholipid binding of the PH domain of the kinase results in its translocation to the plasma membrane and releases an autoinhibitory function of the PH domain. Activation of the kinase is subsequently achieved via its phosphorylation at Thr308 by PDK1 and at Ser473 by PDK2, a postulated kinase. Mutational analysis indicates that constitutive phosphorylation at Ser124 and Thr450 by unknown upstream kinases may be required to prime the enzyme for activation PDK1.

There are thus similarities between the regulation of p70s6k and PKB. The context and location of the essential phosphorylation sites is conserved and each is kept in an inactive conformation, unable to be phosphorylated within the active site until secondary coactivating signals are received by the kinase. In the case of p70s6k, binding of a postulated regulator disrupts the interaction between the N- and C-termini, permitting an ordered set of phosphorylation reactions requiring Thr389 phosphorylation before the final activation by PDK-1. For PKB, the costimulatory binding molecules are PI(3,4,5)P3 and PI(4,5)P2 and this binding allows PDK-1 phosphorylation of Thr308. Phosphorylation of Ser473 is apparently a separate reaction required for maximum enzyme activity. The conserved nature of the hydrophobic motif surrounding Thr389 and Ser473 implies a common upstream kinase (PDK2) may phosphorylate these sites.

In summary, phosphorylation of common sites within the kinase structure is required for activity, and the access of essential upstream kinases is regulated individually by the requirements for binding modula-tory proteins and/or auxiliary phosphorylation reactions.

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Responses

  • vanna
    How multiple phosphorylation allow exquisite regulatory control?
    3 years ago

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