Abstract
Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.
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Acknowledgements
This work was supported by Academy of Finland (CS, SYI), Center of Excellence in Cell stress and Molecular Aging, Ã…bo Akademi (DA), Turku Graduate School for Biomedical Sciences (MS), the Swedish Cancer Society, the Swedish Research Council (DBRM, StratRegen and Project Grant), Karolinska Institute (BRECT, Theme Center in Regenerative Medicine and a Distinguished Professor Award to UL). We are grateful to Helena Saarento for technical assistance. We thank the Cell Imaging Core at Turku Centre for Biotechnology and the proteomics facility of the MRC Protein Phosphorylation Unit, University of Dundee.
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Supplementary information
Supplementary information, Figure S1
Inhibition of aPKC activity attenuates nuclear localization of Myc in Notch1ΔE-myc electroporated chick neural tubes. (PDF 61 kb)
Supplementary information, Figure S2
A) Different Notch constructs expressed in HeLA cells and their immunoreactivity for different antibodies (left cleaved Notch ab, which only recognizes a cleaved Notch1 ICD; right C20 goat, which recognizes an epitope on the Notch intracellular domain, whether it is cleaved or not). (PDF 124 kb)
Supplementary information, Figure S3
Mass spectrometry analysis was performed to identify phosphorylation sites of Notch. (PDF 289 kb)
Supplementary information, Figure S4
Sequence alignment demonstrating the presence or absence of the phosphorylation site in different species and for different Notch isoforms. (PDF 87 kb)
Supplementary information, Figure S5
Nuclear localization of Notch in control and PKCζ expressing cells is inhibited by GSI. (PDF 108 kb)
Supplementary information, Figure S6
Numb overrides the enhancing effects of aPKCζ on Notch signaling. (PDF 80 kb)
Supplementary information, Figure S7
(A) Immunofluorescence images of HeLa cells transfected with WT Notch 1 and Notch1ΔES1791E. (PDF 50 kb)
Supplementary information, Figure S8
(A) Western blot using an antibody against NICD in 293 HEK cells transfected with Notch1ΔE and caPKCζ, Notch1ΔE, Notch1ΔES1791A or Notch1ΔES1769A. (PDF 243 kb)
Supplementary information, Figure S9
Western blot of the nuclear fraction with antibodies against markers for Golgi. (PDF 41 kb)
Supplementary information, Figure S10
PKCζ and PKCι activates Notch signaling and inhibits differentiation of C2C12 myoblasts. (PDF 66 kb)
Supplementary information, Figure S11
(A) Immunofluoresence images of HeLa cells transfected with the Notch1 ΔEGF10-11 mutant in the absence and presence of constitutively active PKCζ. PKCζ shifts the localization of Notch1 ΔEGF10-11 from a perinuclear accumulation in Golgi ER to cytoplasmic vesicles. (PDF 137 kb)
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Sjöqvist, M., Antfolk, D., Ferraris, S. et al. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner. Cell Res 24, 433–450 (2014). https://doi.org/10.1038/cr.2014.34
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DOI: https://doi.org/10.1038/cr.2014.34
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