14-3-3 α and δ Are the Phosphorylated Forms of Raf-activating 14-3-3 β and ζ

Abstract

The 14-3-3 protein family has received considerable attention recently in the literature, because of the finding that beta and zeta isoforms interact with and activate Raf. We had previously shown that these 14-3-3 isoforms also exist as phosphorylated forms in mammalian and avian brain. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly 2-fold. In this report we show by a combination of electrospray mass spectrometry and protein microsequencing that alpha and delta are in vivo post-translationally modified forms of beta and zeta, respectively, and the site of phosphorylation, serine 185, is in a consensus sequence motif for proline-directed kinases.

Isoforms of 14‐3‐3 protein can form homo‐ and heterodimers in vivo and in vitro: implications for function as adapter proteins

“…N and C denote the amino-and carboxy-termini of each subunit. The filled circle indicates the position of the in vivo phosphorylated Ser185 [6] present in the phospho forms offl and ~" 14-3-3. The adjacent shaded portion is the annexin-similarity domain which is also on the surface of 14-3-3 and which has also been proposed to be involved in protein-protein interaction [18].…”

“…Recently the fl and ~" isoforms (which are phosphorylated in vivo at a high stoichiometry in brain [6]) have been shown to associate with (and possibily activate) Raf-1 kinase in the cytosol and at the membrane of mammalian cells and in yeast [11][12][13][14][15]. The r isoform has been shown to bind, and be phosphorylated by, c-Bcr and Bcr-Abl kinases, and may also regulate these proteins [16].…”

“…To date, seven mammalian brain isoforms of 14-3-3 have been described, named a-r/after their respective elution positions on HPLC [4]. Five of these have been sequenced (reviewed in [5]), and the ~ and ~ isoforms are phosphorylated forms of the fl and isoforms respectively [6]. This phosphorylation is at a consensus motif for proline directed (cyclin-dependent) kinases.…”

Four Arabidopsis thaliana 14‐3‐3 protein isoforms can complement the lethal yeast bmh1bmh2 double disruption

“…Two of the isoforms were shown to be a phosphorylated form of other isoforms [36]. From the plant A. thaliana six different cDNAs encoding 14-3-3 protein isoforms have been isolated [37,38].…”

Cross-talk between Aryl Hydrocarbon Receptor and Mitogen-Activated Protein Kinase Signaling Pathway in Liver Cancer through c-raf Transcriptional Regulation

“…It is a downstream effector of ras signaling and a member of the mitogenactivated protein kinase (MAPK) pathway [Ras-Raf-MAPK/ extracellular signal-regulated kinase (ERK) kinase-ERK]. The gene codes for three conserved domains [e.g., ras binding (CR1), activation/inactivation (CR2), and the kinase domain (CR3)] and interacts with an adapter of the 14-3-3 protein family (1). On its dissociation from the 14-3-3 protein, c-raf interacts with ras and undergoes multiple phosphorylation.…”