Interaction of the E1A oncoprotein with Yak1p, a novel regulator of yeast pseudohyphal differentiation, and related mammalian kinases

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.