Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62

Tsai, Li Chun Lisa; Xie, Lei; Doré, Kim; Xie, Li; Rio, Jason C. Del; King, Charles C.; Martinez-Ariza, Guillermo; Hulme, Christopher; Malinow, Roberto; Bourne, Philip E.; Newton, Alexandra C.

doi:10.1074/jbc.m115.676221

Cited by 33 publications

(40 citation statements)

References 56 publications

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“…On CKAR-PB1 p62 , deletion of the pseudosubstrate significantly enhanced basal activity, revealing some autoinhibition by the pseudosubstrate when PKC is bound to p62. This is consistent with our previous study showing that full-length PKC displays ϳ25% of its maximal unrestrained activity on p62 as assessed with a p62-scaffolded CKAR (6). Deletion of the pseudosubstrate had no significant effect on the activity on the Par6 scaffold, revealing that the majority of the PKC bound to Par6 is in the open conformation, as reported previously (7), with the pseudosubstrate tethered away from the substrate-binding cavity.…”

Section: Discussionsupporting

confidence: 92%

“…7A). Analysis of FRET as a measure of translocation (described previously (6)) revealed that insulin stimulation caused translocation of both YFP-p62 and YFP-PKC to CFP-IRS-1, in addition to translocation of YFP-PKC to CFP-p62 (Fig. 7B).…”

Section: Scaffold Proteins Differentially Regulate the Phosphorylatiomentioning

confidence: 67%

“…For experiments with forskolin treatment, cells were treated ϳ24 h prior to imaging and transfected the day before. Kinase activity was monitored via intramolecular FRET of the activity reporters (CKAR or CKAR-PB1), and protein translocation was monitored via intermolecular FRET between the YFP-tagged protein and the CFP-tagged target using methods previously described (6,36).…”

Section: Methodsmentioning

confidence: 99%

“…2A). We have previously used a fusion construct of CKAR tethered to fulllength p62 to measure aPKC activity (6). Because this reporter had a tendency to form large clusters of aggregated p62 within the cells, we fused CKAR to just the PB1 domain of p62 or the PB1 of Par6␣ (which also forms aggregates when expressed in full-length form (7)) as the PB1 domain of each scaffold is the principal surface for aPKC binding (44,45).…”

Section: Pkc Is Constitutively Active On Ckar Substrate Reporter Tethmentioning

confidence: 99%

“…Binding to these scaffolds promotes the open and active conformation of the aPKCs. In the case of p62, an acidic surface unique to its PB1 domain binds the basic pseudosubstrate of aPKCs, tethering it away from the substrate binding cavity to engage aPKC in an active conformation (6). Binding of aPKC to Par6 also displaces the pseudosubstrate to tether aPKC in an open and signalingcompetent conformation (7).…”

mentioning

confidence: 99%

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Protein Scaffolds Control Localized Protein Kinase Cζ Activity

Tobias

Newton²

2016

Journal of Biological Chemistry

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Atypical protein kinase C (aPKC) isozymes modulate insulin signaling and cell polarity, but how their activity is controlled in cells is not well understood. These enzymes are constitutively phosphorylated, insensitive to second messengers, and have relatively low activity. Here we show that protein scaffolds not only localize but also differentially control the catalytic activity of the aPKC PKC, thus promoting activity toward localized substrates and restricting activity toward global substrates. Using cellular substrate readouts and scaffolded activity reporters in live cell imaging, we show that PKC has highly localized and differentially controlled activity on the scaffolds p62 and Par6. Both scaffolds tether aPKC in an active conformation as assessed through pharmacological inhibition of basal activity, monitored using a genetically encoded reporter for PKC activity. However, binding to Par6 is of higher affinity and is more effective in locking PKC in an active conformation. FRETbased translocation assays reveal that insulin promotes the association of both p62 and aPKC with the insulin-regulated scaffold IRS-1. Using the aPKC substrate MARK2 as another readout for activity, we show that overexpression of IRS-1 reduces the phosphorylation of MARK2 and enhances its plasma membrane localization, indicating sequestration of aPKC by IRS-1 away from MARK2. These results are consistent with scaffolds serving as allosteric activators of aPKCs, tethering them in an active conformation near specific substrates. Thus, signaling of these intrinsically low activity kinases is kept at a minimum in the absence of scaffolding interactions, which position the enzymes for stoichiometric phosphorylation of substrates co-localized on the same protein scaffold.

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Section: Discussionsupporting

confidence: 92%

Section: Scaffold Proteins Differentially Regulate the Phosphorylatiomentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Pkc Is Constitutively Active On Ckar Substrate Reporter Tethmentioning

confidence: 99%

mentioning

confidence: 99%

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Protein Scaffolds Control Localized Protein Kinase Cζ Activity

Tobias

Newton²

2016

Journal of Biological Chemistry

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IRS-1 Functions as a Molecular Scaffold to Coordinate IGF-I/IGFBP-2 Signaling During Osteoblast Differentiation

Shen

Rosen

et al. 2016

Journal of Bone and Mineral Research

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Insulin like growth factor I (IGF-I) and insulin like growth factor binding protein-2 (IGFBP-2) function coordinately to stimulate AKT and osteoblast differentiation. IGFBP-2 binding to receptor protein tyrosine phosphatase β (RPTPβ) stimulates polymerization and inactivation of phosphatase activity. Because phosphatase and tensin homolog (PTEN) is the primary target of RPTPβ, this leads to enhanced PTEN tyrosine phosphorylation and inactivation. However RPTPβ inactivation also requires IGF-I receptor activation. The current studies were undertaken to determine the mechanism by which IGF-I mediates changes in RPTPβ function in osteoblasts. IGFBP-2/IGF-I stimulated vimentin binding to RPTPβ and this was required for RPTPβ polymerization. Vimentin serine phosphorylation mediated its binding to RPTPβ and PKCζ was identified as the kinase that phosphorylated vimentin. To determine the mechanism underlying IGF-I stimulation of PKCζ-mediated vimentin phosphorylation, we focused on insulin receptor substrate–1 (IRS-1). IGF-I stimulated IRS-1 phosphorylation and recruitment of PKCζ and vimentin to phospho-IRS-1. IRS-1 immunoprecipitates containing PKCζ and vimentin were used to confirm that activated PKCζ directly phosphorylated vimentin. PKCζ does not contain a SH-2 domain that is required to bind to phospho-IRS-1. To determine the mechanism of PKCζ recruitment we analyzed the role of p62 (a PKCζ binding protein) that contains a SH2 domain. Exposure to differentiation medium plus IGF-I stimulated PKCζ/p62 association. Subsequent analysis showed the p62/PKCζ complex was co-recruited to IRS-1. Peptides that disrupted p62/PKCζ or p62/IRS-1 inhibited IGF-I/IGFBP-2 stimulated PKCζ activation, vimentin phosphorylation, PTEN tyrosine phosphorylation, AKT activation, and osteoblast differentiation. The importance of these signaling events for differentiation was confirmed in primary mouse calvarial osteoblasts. These results demonstrate the cooperative interaction between RPTPβ and the IGF-I receptor leading to a coordinated series of signaling events that are required for osteoblast differentiation. Our findings emphasize the important role IRS-1 plays in modulating these signaling events and confirm its essential role in facilitating osteoblast differentiation.

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