DOI: 10.1042/bj3450297. 14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

10.1042/bj3450297

Crossref journal-article

Portland Press Ltd.

Biochemical Journal (288)

Abstract

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

Bibliography

VAN DER HOEVEN, P. C. J., VAN DER WAL, J. C. M., RUURS, P., VAN DIJK, M. C. M., & VAN BLITTERSWIJK, W. J. (2000). 14-3-3 Isotypes facilitate coupling of protein kinase C-Î¶ to Raf-1: negative regulation by 14-3-3 phosphorylation. Biochemical Journal, 345(2), 297â306.

Authors 5

Paulus C. J. VAN DER HOEVEN (first)
José C. M. VAN DER WAL (additional)
Paula RUURS (additional)
Marc C. M. VAN DIJK (additional)
Wim J. VAN BLITTERSWIJK (additional)

References 0 Referenced 114

None

Dates

Type	When
Created	10 years ago (Aug. 10, 2015, 6:09 p.m.)
Deposited	3 years, 9 months ago (Nov. 23, 2021, 7:44 p.m.)
Indexed	1 week, 1 day ago (Aug. 27, 2025, 12:04 p.m.)
Issued	25 years, 7 months ago (Jan. 10, 2000)
Published	25 years, 7 months ago (Jan. 10, 2000)
Published Online	25 years, 7 months ago (Jan. 10, 2000)
Published Print	25 years, 7 months ago (Jan. 15, 2000)

Funders 0

None

BibTeX

@article{VAN_DER_HOEVEN_2000, title={14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation}, volume={345}, ISSN={1470-8728}, url={http://dx.doi.org/10.1042/bj3450297}, DOI={10.1042/bj3450297}, number={2}, journal={Biochemical Journal}, publisher={Portland Press Ltd.}, author={VAN DER HOEVEN, Paulus C. J. and VAN DER WAL, José C. M. and RUURS, Paula and VAN DIJK, Marc C. M. and VAN BLITTERSWIJK, Wim J.}, year={2000}, month=jan, pages={297–306} }

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