Rb plays a role in survival of Abl-dependent human tumor cells as a downstream effector of Abl tyrosine kinase.

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.

The 14-3-3 protein binds its target proteins with a common site located towards the C-terminus.

The 14-3-3 protein family binds a variety of proteins in cell-signaling pathways, but the structural elements necessary for the ligand binding are poorly understood. Here we demonstrate that the 'box-1' region, which spans residues 171-213 in the eta-isoform and was previously identified as the binding site of 14-3-3 to the phosphorylated tryptophan hydroxylase, plays a critical role in the interaction with many target proteins. Using a series of truncated 14-3-3 mutants, we show that the mutant 167-213 carrying box-1 binds bacurovirus-expressed Raf-1 and Bcr protein kinases to the similar extent as the full-length 14-3-3 in a phosphorylation-dependent manner, while the mutants lacking this region abolish the binding activity. Furthermore, the box-1 region also appears essential for binding of 14-3-3 to more than 40 phosphoproteins found in the brainstem extract. These results suggest that the box-1 region, consisting of helices 7 and 8 in the tertiary structure, is a common structural element whereby the 14-3-3 protein binds many, if not all, target proteins.