Dynamic Features of cAMP-dependent Protein Kinase Revealed by Apoenzyme Crystal Structure

Akamine, Pearl; Madhusudan, M. D.; Wu, Jian; Xuong, Nguyen‐Huu; Eyck, Lynn F. Ten; Taylor, Susan S.

doi:10.1016/s0022-2836(02)01446-8

Cited by 135 publications

(121 citation statements)

References 48 publications

Supporting

Mentioning

113

Contrasting

Unclassified

Order By: Relevance

“…6). The finding that active site inhibitors lock the phosphorylation sites on the C-terminal tail in a phosphatase-resistant conformation is consistent with structural studies of PKA that showed that the C-terminal tail is highly ordered when inhibitor is bound and highly disordered in the apo structure (39,40). It is interesting to note that occupancy of the active site by ATP analogues such as Bis I expels the autoinhibitory pseudosubstrate from the substrate-binding cavity (32), an event that, in itself, increases the phosphatase sensitivity of PKC by 2 orders of magnitude (7).…”

Section: Discussionsupporting

confidence: 84%

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Gould

Antal

Reyes

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.PKC isozymes comprise a family of multidomain proteins that are under exquisite conformational control. Two major mechanisms control the conformation of PKC family members: phosphorylation and second messenger-dependent membrane binding (1, 2). First, newly synthesized enzymes undergo a series of ordered phosphorylations that lock the enzyme into a stable, catalytically competent, and autoinhibited species (3, 4). This species is maintained in an autoinhibited conformation by a pseudosubstrate segment that blocks the substrate-binding cavity, a conformation that also protects the priming sites of PKC from dephosphorylation. The inactive species are localized throughout the cell, often tethered to scaffold proteins (5). This processing by phosphorylation is constitutive and required to protect PKC from degradation; unphosphorylated protein is rapidly degraded (1). Second, binding to lipid second messengers allosterically controls the enzyme by facilitating the release of the autoinhibitory pseudosubstrate segment from the substrate-binding cavity (6). Thus, this conformational transition is acutely controlled by activation of receptors that signal using diacylglycerol as the second messenger. The membranebound conformation has an increased sensitivity to phosphatases by 2 orders of magnitude (7), and prolonged activation, as occurs with phorbol esters (functional analogues of diacylglycerol), results in the dephosphorylation and subsequent degradation of PKC (8). Thus, phosphorylation converts newly synthesized PKC into a stable, degradation-resistan...

show abstract

Section: Discussionsupporting

confidence: 84%

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Gould

Antal

Reyes

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…These turn out to be PKA structures of the APO form (e.g., 1J3H and 1SYK), which undergoes a significant conformational change compared with the ligand-bound form. 20,21 Not surprisingly, structures with RMSD's between 1 Å and 2 Å (23% of KKB structures) have the highest sequence similarity to PKA, between 25% and 50%. The majority of the kinases have RMSD's in the 2.0-2.5 Å (43%) and 2.5-3.0 Å (26%) range.…”

Section: Resultsmentioning

confidence: 99%

An enriched structural kinase database to enable kinome‐wide structure‐based analyses and drug discovery

et al. 2010

View full text Add to dashboard Cite

The development of a kinase structural database, the kinase knowledge base (KKB), is described. It covers all human kinase domain structures that have been deposited in the Protein Data Bank. All structures are renumbered using a common scheme, which enables efficient crosscomparisons and multiple queries of interest to the kinase field. The common numbering scheme is also used to automatically annotate conserved residues and motifs, and conformationally classify the structures based on the DFG-loop and Helix C. Analyses of residue conservation in the ATP binding site using the full human-kinome-sequence alignment lead to the identification of a conserved hydrogen bond between the hinge region backbone and a glycine in the specificity surface. Furthermore, 90% of kinases are found to have at least one stabilizing interaction for the hinge region, which has not been described before.

show abstract

“…Interconversions between "open" and "closed" domain conformations have been noted in other enzymes, including protein kinases (23,24). In the cAMPdependent protein kinase catalytic subunit (PKA-C), the X-ray structure of the apo form shows an open conformation, whereas a binary complex with Mg 2+ -AMP-PNP shows a closed conformation formed by rotation of the N-terminal domain and closure of the catalytic cleft around the nucleotide (25,26). NMR relaxation measurements of backbone amides in the nucleotide-PKA-C binary complex show global exchange behavior in residues lining the catalytic core (27,28), which are absent in apo PKA-C, and have been interpreted as an equilibrium shift to a closed conformation.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation releases constraints to domain motion in ERK2

Xiao¹,

Lee

Latham³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

143

View full text Add to dashboard Cite

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13 C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k ex ≈ 300 s −1 (k AB ≈ 240 s −1 /k BA ≈ 60 s −1 ) and p A /p B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N-and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k ex ≈ 500 s −1 (k AB ≈ 15 s −1 /k BA ≈ 485 s −1 ) and p A /p B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.T he MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phosphoThr and phospho-Tyr residues and basic residues in the N-and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogenexchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7-9) led to a model where conformational mobility at the hinge linking the N-and C-terminal domains is increased by phosph...

show abstract

Dynamic Features of cAMP-dependent Protein Kinase Revealed by Apoenzyme Crystal Structure

Cited by 135 publications

References 48 publications

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

An enriched structural kinase database to enable kinome‐wide structure‐based analyses and drug discovery

Phosphorylation releases constraints to domain motion in ERK2

Contact Info

Product

Resources

About