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

Abstract

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.

Figure 1

Map of the major E1A proteins and conserved regions. The two major products of E1A are 289 and 243 residues in length and differ only by the presence of an additional 46 amino acids unique to the larger. The C-terminal region of E1A corresponding to aa 187–289 was expressed as a fusion with the Gal4p DBD, LexA DBD, or GFP in this study.

Figure 2

Effect of the C-terminal domain of E1A on yeast pseudohyphal growth. Wild-type diploid yeast of the Σ1278b background (JMY38 a/α) were transformed with a control vector; with vectors expressing the C-terminal region of E1A fused to the Gal4p DBD (A), LexA DBD (B), or GFP (C); or with vectors expressing the HPV 16 E7 protein similarly fused to the Gal4p (A) or LexA DBD (B). Transformed yeast were grown on SLAD medium for 2 d at 30°C and photographed.

Figure 3

Induction of pseudohyphal growth by E1A mutants containing small deletions in the C-terminal region. Wild-type diploid yeast of the Σ1278b background (JMY38 a/α) were transformed with a control vector, a vector expressing the C-terminal region of E1A fused to the Gal4p DBD, or similar vectors expressing mutant forms of the C-terminal region of E1A containing the indicated amino acid deletions. Transformed yeast were grown on SLAD medium for 2 d at 30°C and photographed.

Figure 4

Ability of the C-terminal domain of E1A to induce pseudohyphal growth in yeast mutated for components of the MAPK signal transduction pathway. Diploid yeast strains with homozygous disruptions of STE7 (L5986) (A), KSS1 (L6278) (B), or STE12 (L5987) (C) were transformed with either a control vector without E1A (left column) or a vector expressing the C-terminal region of E1A (middle column). Transformants were grown on SLAD medium for 2 d at 30°C and photographed. Diploid yeast of the same strains transformed only with complementing vectors expressing STE7 (A), KSS1 (B), or STE12 (C) are shown in right column.

Figure 5

Ability of the C-terminal domain of E1A to induce pseudohyphal growth in yeast with alterations in the cAMP/PKA signal transduction pathway. The indicated transformants were grown on SLAD medium for 2 d at 30°C and photographed (A–E). Wild-type diploid yeast (JMY38 a/α) were transformed with vectors overexpressing PDE1 (A), PDE2 (B), or BCY1 (C) and either a control vector without E1A (left column) or a vector expressing the C-terminal region of E1A (middle column). Yeast of the same strain transformed with two empty control vectors are shown in the right column. (D–F) Diploid yeast strains with homozygous disruptions of TPK2 (XPY5) (D), FLO8 (XPY95) (E), or FLO11 (XPY107) (F) were transformed with either a control vector without E1A (left column) or a vector expressing the C-terminal region of E1A (middle column). (D) Cells of strain XPY95 transformed with a vector expressing TPK2 are shown in the right column.

Figure 6

Dependence of the E1A effect on Phd1p. A diploid yeast strain homozygously disrupted for PHD1 (L6213) was transformed with a control vector without E1A, a vector expressing the C-terminal region of E1A, or a vector expressing PHD1. Transformants were grown on SLAD medium for 2 d at 30°C and photographed.

Figure 7

Interaction of yeast Yak1p with the E1A C-terminal deletion mutants in the yeast two-hybrid assay. Yeast strain L40 was transformed with expression vectors for the indicated LexA-E1A fusions and expression vectors for YAK1 or CtBP fused to a transcriptional activation domain. Transformed yeast were streaked on nonselective plates (+histidine) and selection plates (−histidine) and allowed to grow at 30°C for 3 d. For each E1A mutant used as “bait,” the indicated numbers are inclusive and refer to the amino acid residues deleted with respect to the 289R E1A protein.

Figure 8

Coprecipitation of E1A with mammalian homologs of Yak1p. Purified recombinant rat GST-Dyrk1A or human GST-Dyrk1B was incubated with yeast extracts containing either the 243R or 289R E1A proteins (see MATERIALS AND METHODS). The GST-Dyrk protein complexes were then pulled down with glutathione-Sepharose beads and the proteins were analyzed by SDS-PAGE and imunoblotted with a monoclonal antibody against E1A. The empty GST vector was used as a negative control, and the input levels of E1A were as shown.

Figure 9

Effect of E1A on kinase activity of recombinant rat Dyrk1A. (A) Effect of E1A on autophosphorylation. GST-Dyrk1A was incubated with GST-E1A 243R in phosphorylation buffer for 2 h on ice. Following the addition of [γ-³²P]ATP, samples were incubated for 30 min at 30°C, resolved on 15% SDS-PAGE, and analyzed using a Molecular Dynamics phosphoImager. Lane 1 contains 12 μg of GST-Dyrk1A. Lanes 2–6 contain 12 μg of GST-Dyrk1A with 2, 4, 6, 8, or 12 μg of GST-E1A 243R. Lane 7 contains 12 μg of GST-E1A 243R. (B) Effect of E1A on phosphorylation of histone H3. The assay was performed as described in A except for the presence of histone H3. Fold changes in kinase activity are indicated below each lane.

Figure 10

Effect of Yak1p overexpression on yeast pseudohyphal growth. Wild-type diploid yeast (JMY38 a/α) were transformed with a control vector or a vector expressing full-length YAK1 under the transcriptional control of the ADH1 promoter. Transformed yeast were grown on SLAD medium for 2 d at 30°C and photographed.

Figure 11

Genetic analysis of the role of Yak1p in regulating pseudohyphal growth. Wild-type diploid yeast (MLY61 a/α) or yeast disrupted for YAK1 (DSY1 a/α) were transformed with a control vector (A), or with vectors expressing RAS2^VAL19 (B), STE11-4 (C), STE12 (D), TEC1 (E), TPK2 (F), PHD1 (G), or the C terminus of E1A (H). Transformed cells were then transferred to SLAD plates, allowed to grow for 2 d at 30°C, and photographed.