Cation Diffusion Facilitator Proteins Modulate Raf-1 Activity

Abstract

The Ras-extracellular signal-regulated kinase (ERK) cascade is a critical intracellular signaling pathway that regulates growth, survival, and differentiation. Previous work established that Ras-GTP binds to, and facilitates the activation of, the protein kinase Raf-1. Recently, it was demonstrated that the cation diffusion facilitator (CDF) proteins are involved in Ras-ERK signaling by use of a Caenorhabditis elegans genetic screen that identified suppressors of activated Ras. In the current work, we demonstrate that CDF proteins may function downstream of Ras, but upstream of Raf-1 in Xenopus oocytes. We also show that the C. elegans protein CDF-1 and its mammalian homologue ZnT-1 bind to the amino-terminal regulatory portion of Raf-1 and promote the biological and enzymatic activity of Raf-1. Furthermore, we show that Zn 2؉ inhibits Raf-1 binding to ZnT-1. We propose a model in which CDF protein binding facilitates Raf-1 activation.The Raf-1 protein kinase plays an important role in signal transduction in eukaryotic cells (1-3). Raf-1 is a member of a multigene family that includes A-Raf and B-Raf. Raf family members regulate cell proliferation and differentiation, and are also involved in the pathogenesis of many forms of human cancer. A recent study found that mutations in B-Raf are present in 66% of human melanomas, highlighting the importance of this gene family (4).When active, Raf-1 phosphorylates and activates MEK1, 1 a protein threonine and tyrosine kinase that, in turn, phosphorylates and activates the mitogen-activated protein kinase (MAPK) family members ERK1 and -2 (hereafter called ERK) (5-7). Raf-1 activation is a highly complex and incompletely understood process. Although the three-dimensional x-ray crystallographic structure of Raf-1 has not been solved, a widely accepted model is that that amino-terminal portion of Raf-1 folds over the carboxyl-terminal half to inhibit its kinase activity (8). 14-3-3 dimers may stabilize the inactive conformation of Raf-1 by interacting simultaneously with phosphoserine 259 and phosphoserine 621 of inactive Raf-1 (9 -13). Inactive Raf-1 is also bound to several heat shock proteins that may prevent the proteasome-mediated degradation of Raf-1 and facilitate its cytoplasmic localization (14, 15).The Ras family of small GTPases plays a key role in the activation of . When bound to GTP, Ras binds to two domains on Raf-1, the Ras-binding domain comprising amino acids 51-131, and the cysteine-rich domain (CRD) comprising amino acids 139 -184 (19 -21). Recently, Morrison's group demonstrated that protein phosphatase 2A dephosphorylates phosphoserine 259 of Raf-1 to release 14-3-3 and promote Ras-GTP binding to the CRD in growth factor-stimulated cells (22). By binding to Raf-1, Ras also promotes the plasma membrane localization of Raf-1. Therefore, Ras-GTP facilitates Raf-1 translocation and activation. However, Ras-GTP is not sufficient in most cases to fully activate Raf-1. First, several phosphorylation events occur at the plasma membrane, which facilitate activ...

Zinc-Dependent Suppression of TNF-α Production Is Mediated by Protein Kinase A-Induced Inhibition of Raf-1, IκB Kinase β, and NF-κB

“…A different mechanism for zinc-mediated down-regulation of Raf-1 activity has been found in Xenopus oocytes. In this study, the kinase activity of Raf-1 is promoted by binding to the zinc export protein ZnT-1, and the addition of zinc inhibits the binding of Raf-1 to ZnT-1 (31). Such a mechanism of Raf-1 inhibition by zinc can be excluded in monocytes, because inhibition of sGC (9) or PKA antagonized the inhibitory effect of zinc on TNF-␣ secretion, providing evidence for an involvement of cyclic nucleotide signaling.…”

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

“…These regions may therefore contain the key structural elements that enable the interaction of ZnT-1 with other proteins, including the ␤-subunit. The notion that the underlying mechanism for ZnT-1 activity involves protein-protein interaction has been shown previously in other systems (45,65). Jirakulaporn et al (45) reported that the ZnT-1 C-terminal segment binds to N-Raf (cytoplasmic protein) and by doing so activates the extracellular signal-regulated kinase-mitogen-activated protein kinase cascade (ERK-MAPK).…”

“…The notion that the underlying mechanism for ZnT-1 activity involves protein-protein interaction has been shown previously in other systems (45,65). Jirakulaporn et al (45) reported that the ZnT-1 C-terminal segment binds to N-Raf (cytoplasmic protein) and by doing so activates the extracellular signal-regulated kinase-mitogen-activated protein kinase cascade (ERK-MAPK). Moreover, their study also showed that high levels of free zinc impair the interaction between ZnT-1 and Raf-1, which may imply a conformational change of ZnT-1 in the presence of high zinc concentrations.…”

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

“…Taken together with our results, this suggests that the cytosolic C-terminal tail of CDF/ZnT proteins is important in determining their oligomeric partner molecules. The cytosolic C-terminal tail of CDF/ZnT proteins has been shown to be important for binding of both zinc and a signal transduction molecule (12,13,37). Further studies may reveal additional functions for the C-terminal region.…”