Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Swanson, Carter J.; Ritt, Michael; Wang, William Yang; Lang, Michael J.; Narayan, Arvind; Tesmer, John J.G.; Westfall, Margaret V.; Sivaramakrishnan, Sivaraj

doi:10.1074/jbc.m113.534750

Cited by 22 publications

(44 citation statements)

References 66 publications

(96 reference statements)

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“…This finding is consistent with the dominant role of the pseudosubstrate in PKC autoinhibition (20). (14,15), which separates the catalytic domain of PKC␣ and a variety of short peptides Յ14 residues in length (Fig. 2).…”

Section: Resultssupporting

confidence: 77%

“…3B) (19). Human PKC␣ cDNA was purchased from Open Biosystems as described previously (15). PKA and PKC␣ constructs were cloned using PCR (Expand High Fidelity PCR System, Sigma) or site-directed mutagenesis (Pfu-Turbo, Agilent) into pBiex1 (Novagen) Sf9 expression plasmid vector.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, only a few crystal structures have thus far been reported for the kinase catalytic domain bound to substrate (4). Here, we instead use a recently developed approach to compare weak ( M) interactions between any two proteins or protein domains (14,15). We apply this approach to directly compare the binding affinities of a kinase catalytic domain for a variety of weak binding substrate peptides.…”

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confidence: 99%

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Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

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Section: Resultssupporting

confidence: 77%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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“…Swanson et al [33] showed evidence that PKCβII forms homo-dimers upon activation and that the dimers maintain PKC in an active conformation. Both C1 domains and the C2 domain contribute to this interaction, but the C-terminal tail is absolutely necessary for dimer formation.…”

Section: Regulation By Conformational Changesmentioning

confidence: 99%

“…Both C1 domains and the C2 domain contribute to this interaction, but the C-terminal tail is absolutely necessary for dimer formation. In fact, phosphorylation of the C-terminal tail is critical in maintaining PKC in a closed, inactive conformation, as lack of phosphorylation at these sites leads to PKC remaining in an open, exposed conformation [30] and increases basal dimerization [33]. Therefore, PKC undergoes conformational changes both during its maturation and during its activation in order to finely tune its activity.…”

Section: Regulation By Conformational Changesmentioning

confidence: 99%

Tuning the signalling output of protein kinase C

Antal

Newton

2014

Biochemical Society Transactions

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Precise control of the amplitude of protein kinase C signaling is essential for maintaining cellular homeostasis, and disruption of this control leads to pathophysiological states such as cancer and neurodegeneration. This amplitude is precisely tuned by multiple inputs that regulate the amount of protein kinase C in the cell, its ability to sense its allosteric activator diacylglycerol, and protein scaffolds that coordinate access to substrates. Key to regulation of the signaling output of most protein kinase C isozymes is the ability of cytosolic enzyme to respond to the membrane-embedded lipid second messenger, diacylglycerol, in a dynamic range that prevents signaling in the absence of agonists but allows efficient signaling in response to small changes in diacylglycerol. This review discusses the regulatory inputs that control the spatiotemporal dynamics of protein kinase C signaling, focusing on conventional and novel PKC isozymes.

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The tumor promoter‐activated protein kinase Cs are a system for regulating filopodia

et al. 2017

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Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, β, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter‐stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/β pseudosubstrate sequence, and an inhibitor of T‐cell protein tyrosine phosphatase (TC‐PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter‐treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ‐mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady‐state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long‐term “memory” of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

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Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Cited by 22 publications

References 66 publications

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Tuning the signalling output of protein kinase C

The tumor promoter‐activated protein kinase Cs are a system for regulating filopodia

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