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

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.

Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

“…Most studies of the role of 14-3-3 in cellular regulation have focused on changes in target protein phosphorylation as the initial regulatory event; however, 14-3-3 proteins have been shown to be phosphorylated in animal systems (37,38) and in plants (39)(40)(41). Phosphorylation of 14-3-3 is another way in which these proteins are regulated (29), and in animal systems, a few kinases reportedly phosphorylate 14-3-3.…”

14-3-3 Proteins Regulate Protein Kinase A Activity to Modulate Growth Cone Turning Responses

“…The 14-3-3 proteins are adapter proteins that interact with specific phospho-serine and phospho-threonine motifs within a number of binding proteins and consequently regulate diverse cellular processes including cell survival, metabolism, proliferation and protein trafficking (van Heusden, 2005;Garbe and Bashaw, 2004;Clandinin and Zipursky, 2002;Jin et al, 2004). The 14-3-3 proteins function as homodimers and heterodimers to control the spatial and temporal activity of substrate proteins by regulating subcellular localization (Muslin and Xing, 2000;Nagata-Ohashi et al, 2004), binding partner proximity (Jones et al, 1995;Berruti, 2000;Van Der Hoeven et al, 2000) and by inducing conformational changes that can affect interactions between binding proteins and their substrates (Roy et al, 1998). In mammals, there are seven 14-3-3 isoforms, designated ␤ or ␣ (␤,␣), , , ␥, or ( , ), or ␦ ( ,␦), and /stratifin ( ) (Bridges and Moorhead, 2005).…”

Identification of 14-3-3ζ as a Protein Kinase B/Akt Substrate

“…Additionally, phosphorylation of 14-3-3 proteins impairs their interaction with protein kinase C (14). Three phosphorylation sites on 14-3-3, Ser-58, Ser-184, and Thr-232, have been identified (5).…”