Abstract
Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.
Highlights
Following stimulation of membrane receptors, the Raf-1 kinase is activated and transduces signals to several signaling pathways [1,2,3]
Studies using overexpression systems have demonstrated that Raf-1 can be activated both by tyrosine phosphorylation mediated by members of the Src kinase family [7, 8] and by serine phosphorylation, mediated by protein kinase C [9]
It was recently shown that the Raf-1 activating phosphorylation at Tyr340, which is mediated by members of the Src kinase family, is triggered by CD4 cross-linking in T cells [12] and by Fc␥RI cross-linking in myeloid cells [13]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF123704. CAMP-dependent protein kinase A [10] and an unidentified G-protein-coupled tyrosine phosphatase down-regulate the activity of Raf-1 [11]. The GTP-charged form of Ras binds directly to the aminoterminal conserved region 1 which includes the Ras-binding domain and the cysteine-rich domain of Raf-1 (28 –31) This interaction causes translocation of Raf-1 from the cytosol to the plasma membrane where Raf-1 activation takes place [24, 25]. The nature of the membrane events and the macromolecules involved in Raf-1 activation have not been well defined to date One such mechanism, may involve binding of Raf-1 to the oligomer forming adaptor protein 14-3-3 [32]. Overexpression of Tvl-1 in COS-1 cells enhanced the EGF1-induced Raf-1 activation. These findings suggest that Tvl-1 may contribute to the regulation of Raf-1 activation as well as to the transduction of Raf-1 signals
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