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Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions.

Rekha N, Srinivasan N - BMC Struct. Biol. (2003)

Bottom Line: In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed.The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported.The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India. rekha@mbu.iisc.ernet.in

ABSTRACT

Background: Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (alpha) and two identical regulatory (beta) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2alpha in isolation. The present work explores the structural basis of this feature using computational analysis and modeling.

Results: We have initially built a model of PKCK2alpha bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in beta turn, alpha helix, or 3(10) helices. This type of conformation is observed and accommodated well by PKCK2alpha in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2alpha phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2alpha complex. PKCK2beta undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme.

Conclusion: Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2alpha, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

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Structural model of binding of PKCK2α (blue) and PKCK2β (brown) during autophosphorylation. The sidechains of catalytic base, Asp156, in the catalytic subunit and the phosphorylation site, Ser 2, in the PKCK2β are also shown. (Figure generated using SETOR [44])
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Figure 4: Structural model of binding of PKCK2α (blue) and PKCK2β (brown) during autophosphorylation. The sidechains of catalytic base, Asp156, in the catalytic subunit and the phosphorylation site, Ser 2, in the PKCK2β are also shown. (Figure generated using SETOR [44])

Mentions: PKCK2 holoenzyme is known to phosphorylate the PKCK2β subunit [1,36]. This auto-phosphorylation down regulates the PKCK2 activity. The phosphorylation site in the β-subunit is at the N-terminal (Ser 2). This site is visible in the electron density corresponding to one of the β-chains in the crystal structure of holoenzyme [26]. In our model Ser 2 has been placed at a hydrogen bonding orientation from the catalytic base Asp 156 of the α-subunit. Further, the downstream acidic charges are interacting with basic substrate binding determinants. The model of PKCK2α binding to PKCK2β in substrate binding position is generated as discussed in Methods and is shown in figure 4. The charges on the surface of the β-subunit compliments with the surface charges of α-subunit, with positive charges on α-subunit and negative charges on the β-subunit as shown in figure 5.


Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions.

Rekha N, Srinivasan N - BMC Struct. Biol. (2003)

Structural model of binding of PKCK2α (blue) and PKCK2β (brown) during autophosphorylation. The sidechains of catalytic base, Asp156, in the catalytic subunit and the phosphorylation site, Ser 2, in the PKCK2β are also shown. (Figure generated using SETOR [44])
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161795&req=5

Figure 4: Structural model of binding of PKCK2α (blue) and PKCK2β (brown) during autophosphorylation. The sidechains of catalytic base, Asp156, in the catalytic subunit and the phosphorylation site, Ser 2, in the PKCK2β are also shown. (Figure generated using SETOR [44])
Mentions: PKCK2 holoenzyme is known to phosphorylate the PKCK2β subunit [1,36]. This auto-phosphorylation down regulates the PKCK2 activity. The phosphorylation site in the β-subunit is at the N-terminal (Ser 2). This site is visible in the electron density corresponding to one of the β-chains in the crystal structure of holoenzyme [26]. In our model Ser 2 has been placed at a hydrogen bonding orientation from the catalytic base Asp 156 of the α-subunit. Further, the downstream acidic charges are interacting with basic substrate binding determinants. The model of PKCK2α binding to PKCK2β in substrate binding position is generated as discussed in Methods and is shown in figure 4. The charges on the surface of the β-subunit compliments with the surface charges of α-subunit, with positive charges on α-subunit and negative charges on the β-subunit as shown in figure 5.

Bottom Line: In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed.The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported.The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India. rekha@mbu.iisc.ernet.in

ABSTRACT

Background: Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (alpha) and two identical regulatory (beta) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2alpha in isolation. The present work explores the structural basis of this feature using computational analysis and modeling.

Results: We have initially built a model of PKCK2alpha bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in beta turn, alpha helix, or 3(10) helices. This type of conformation is observed and accommodated well by PKCK2alpha in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2alpha phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2alpha complex. PKCK2beta undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme.

Conclusion: Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2alpha, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

Show MeSH