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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.
The human adenovirus early region 1A (E1A) gene encodes two major proteins of 289 and 243 residues, which differ only by the presence of an internal sequence of 46 amino acids unique to the larger protein (Figure (Figure1).1). Comparison of the E1A sequences of a number of adenovirus serotypes has identified three regions of sequence conservation (Kimelman et al., 1985 ; van Ormondt et al., 1986 ), designated conserved regions 1, 2, and 3. Exon 2 of E1A encodes 104 amino acids that are not highly conserved between various adenovirus serotypes. Nevertheless, this C-terminal region of E1A is required for immortalization by E1A and transformation of rodent cells in cooperation with the products of the adenovirus E1B oncogene (Subramanian et al., 1991 ). In contrast to its function with the E1B proteins, the C terminus of E1A acts to repress transformation in cooperation with ras and blocks the invasive and metastatic properties of ras-transformed cells (Subramanian et al., 1989 ; Douglas et al., 1991 ; Linder et al., 1992 ; Boyd et al., 1993 ). The only known protein target of the C terminus of E1A is CtBP (Schaeper et al., 1995 ), a transcriptional corepressor (Criqui-Filipe et al., 1999 ; Furusawa et al., 1999 ; Meloni et al., 1999 ; Sewalt et al., 1999 ). Disruption of the interaction of E1A with CtBP enhances the ability of E1A to cooperate with activated ras to transform cells and increases the tumorigenic and metastatic capacity of these transformed cells. However, the interaction of CtBP with E1A may not completely account for these activities, because E1A mutants with deletions of other regions within the C terminus show similarly increased metastatic and tumorigenic properties, yet still retain the ability to interact with CtBP (Boyd et al., 1993 ; Schaeper et al., 1995 ).
Progress in elucidating the mechanisms by which the C-terminal region of E1A functions to modulate ras-induced transformation, tumorigenesis, and metastasis has been hampered by the technical difficulties associated with genetic analysis in mammalian cells. In contrast, powerful tools are available for molecular genetic analysis in the yeast Saccharomyces cerevisiae. Furthermore, expression of a foreign gene in yeast can provide an attractive means of studying gene function because many important biological processes are conserved between higher eukaryotes and yeast. Indeed, our recent studies have established that E1A interacts with conserved gene and growth regulatory pathways in yeast (Miller et al., 1995 , 1996 ), providing a basis for more detailed investigations of the molecular mechanisms by which E1A reprograms cell growth and development.
In this study, we have examined the effects of the C-terminal domain of E1A on Ras2p-regulated growth control in S. cerevisiae. In this dimorphic yeast, the product of RAS2, the yeast homolog of mammalian h-ras, controls mitogen-activated protein kinase (MAPK) and cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathways to regulate pseudohyphal/filamentous differentiation in diploid cells and invasion of the growth matrix by haploid cells (Gimeno et al., 1992 ; Liu et al., 1993 ; Mösch et al., 1996 ; Cook et al., 1997 ; Madhani et al., 1997 ; Robertson and Fink, 1998 ; Pan and Heitman, 1999 ). Expression of the C-terminal domain of E1A induced strong pseudohyphal growth in diploid yeast, which was independent of both the MAPK and cAMP/PKA pathways and thus functions through a novel mechanism. Using the yeast two-hybrid system, we identified an interaction between the C terminus of E1A and yeast Yak1p, a dual-specificity serine/threonine kinase (Garrett et al., 1991 ) that functions as a negative regulator of growth (Garrett and Broach, 1989 ).
Yeast strains used in these experiments are listed in Table Table1.1. All strains used for pseudohyphal growth assays are derived from the Σ1278b background (Grenson et al., 1966 ). Yeast culture media was prepared and yeast genetic manipulations were performed using standard techniques (Adams et al., 1998 ). Synthetic low-ammonia dextrose (SLAD) medium for pseudohyphal growth assay was prepared as described (Gimeno et al., 1992 ).
Plasmids used in this study are listed in Table Table22 and the construction of those plasmids novel to this report are summarized below. Yeast expression vector pAS1U was constructed from pAS1 (Durfee et al., 1993 ) by subcloning the XbaI-NaeI fragment of pAS1 into the same sites of pRS426 (Christianson et al., 1992 ). The C-terminal domain of E1A (amino acids [aa] 187–289) was expressed as a fusion with the Gal4p DBD (aa 1–147) by subcloning an EcoRI-BamHI fragment from pMA-Ex2 (Schaeper et al., 1995 ) into pAS1U. The C-terminal domain of E1A was expressed as fusion with the LexA DBD from pSH2-X2, which was constructed by subcloning the same EcoRI-BamHI fragment into pSH2–1 (Hanes and Brent, 1989 ). The E7 protein of HPV16 was polymerase chain reaction (PCR) subcloned as an EcoRI-SalI fragment from pATH11-E7 (Carter et al., 1991 ) into pAS1U and pSH2–1 to make pAS1U-E7 and pSH2-E7, respectively. pRS423-LexA was constructed from pEG202 (OriGene Technologies, Rockville, MD) by subcloning the SphI-SalI fragment of pEG202 into the same sites of pRS423-ADH (Mumberg et al., 1995 ). pBait was constructed by moving a PvuII fragment from pRS423-LexA into the same sites of YEplac181 (Gietz and Sugino, 1988 ). The region encoding the C terminus of E1A was cloned as an EcoRI-BamHI fragment from pMA-Ex2 (Schaeper et al., 1995 ) into pBait to make pBait-X2. To construct LexA DBD fusions with E1A proteins with deletions in the C-terminal region of E1A, EcoRI-BamHI or EcoRI-SalI fragments from the previously described series of pMA-Ex2 deletion mutant plasmids (Schaeper et al., 1995 ) were subcloned into corresponding sites of pBait. pGFP was constructed by subcloing an NheI-HindIII fragment of pEGFP-C1 (Clontech, Palo Alto, CA) and the self-complementary oligos JMO-44 (5′-AGCTTCTGAATTCCCGGGGATCCCTGCAG-3′) and JMO-45 (5′-TCGACTCCAGGGATCCCCGGGAATTCAGA-3′) into the SpeI-SalI sites of pRS426-ADH (Mumberg et al., 1995 ). pGFP-X2 was constructed by subcloning an EcoRI-BamHI fragment encoding the C terminus of E1A from pMA-Ex2 into the same sites of pGFP. pRS313-STE11-4 was constructed by insertion of a BamHI-SalI fragment containing STE11-4 from plasmid pSL1509 (Stevenson et al., 1992 ) into pRS313 (Sikorski and Hieter, 1989 ). The two-hybrid prey plasmid pRS424-VP16 was constructed by subcloning a NheI-SalI fragment from pVP16 (Clontech) into the SpeI-SalI sites of pRS424-GAL1 (Mumberg et al., 1995 ). Drosophila CtBP was inserted into pRS424-VP16 as a BamHI fragment from plasmid h-C28 (Poortinga et al., 1998 ). The sequences encoding the 289R and 243R E1A proteins were PCR amplified and subcloned as EcoRI-XhoI fragments into pRS423-GAL1 to construct pRS423GAL-289R and pRS423GAL-243R, respectively (Mumberg et al., 1995 ). The same fragments were subcloned into pGEX-4T1 (Amersham Pharmacia Biotech, Baie d'Urfé, Québec, Canada) to construct pGST-289R and pGST-243R. pRS426-Yak1 was constructed by PCR amplification of the YAK1 coding region from yeast genomic DNA and subcloning it into BamHI and XhoI sites of pRS426-ADH (Mumberg et al., 1995 ).
Growth assays for filament formation in colonies of diploid cells were performed as described previously (Gimeno et al., 1992 ). Briefly, diploid yeast were transformed with expression vectors by using lithium acetate (Adams et al., 1998 ). Single colonies of transformed yeast were patched onto SLAD plates and scored for pseudohyphal filament formation after 2 d of growth at 30°C. Representative single colonies were directly photographed with a Leitz Diaplan light microscope equipped with a 40× long working distance objective and a Sony PowerHAD 3CCD color video camera using Northern Eclipse Imaging Analysis software (Empix Imaging, Mississauga Ontario, Canada).
The plasmids pIL30-URA3 or pIL30-HIS3 (Laloux et al., 1994 ), which contain the LacZ gene under the transcriptional control of a filamentation response element (Mösch et al., 1996 ), were used to monitor MAPK-mediated activation of transcription during pseudohyphal growth. To examine the effect of the C-terminal domain of E1A on MAPK signaling, diploid yeast of the Σ1278 background were transformed with a reporter plasmid together with plasmids expressing the E1A fusion protein. β-Galactosidase assays were prepared as previously described (Mösch et al., 1996 ). Protein concentrations from the clarified extracts were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as a standard. β-Galactosidase activity (nmol/min/mg protein) was calculated using the following equation (OD420 × 1.7)/(0.0045 × protein concentration [mg/ml] × extract volume [ml] × time [min]) (Adams et al., 1998 ).
Yeast strain L40 (Invitrogen, Carlsbad, CA) was transformed as described by Gietz et al. (1997) with pBait-X2 and a yeast genomic library in plasmid pJG4–5 (OriGene Technologies) kindly provided by Dr. M. Christman (University of Virginia, Charlottesville, VA). About 2 × 107 yeast transformants were screened for the ability to grow in the absence of histidine.
Vectors expressing GST fusions of either E1A, DYRK1A, or DYRK1B (Kentrup et al., 1996 ) were introduced into BL21 E. coli cells and recombinant fusion proteins were prepared and purified using glutathione-Sepharose as described by the manufacturer (Amersham Pharmacia Biotech, Baie d'Urfé, Québec, Canada).
Yeast cells transformed with pRS423GAL-289R and pRS423GAL-243R, which express the larger or smaller major E1A protein under the control of the GAL1 promoter, were grown in synthetic complete selection medium containing glucose until the cell density reached 0.8. Cells were pelleted, washed with water, and resuspended in selection medium containing galactose for 6 h. Cells were collected by centrifugation, resuspended in 100 mM Tris-HCl buffer (pH7.5) containing 1 mM dithiothreitol and 20% glycerol, and disrupted with glass beads by vortexing. Glass beads and cell debris were removed by centrifugation at 4°C. The supernatant was used for in vitro interaction assays with E. coli-produced GST-DYRK fusions. Purified GST-DYRK fusion proteins were incubated with yeast extracts containing E1A overnight at 4°C in phosphate-buffered saline buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) for protein complex formation. GST-DYRK protein complexes were purified by affinity absorption to glutathione-Sepharose by using standard procedures, separated by SDS-PAGE, and E1A was detected by Western blotting with the E1A monoclonal antibody M73 (Harlow et al., 1985 ).
To detect the effect of E1A on DYRK phosphorylation activity, GST-DYRK proteins were incubated with GST-E1A fusion protein in phosphorylation buffer (33 mM HEPES, 6.6 mM manganese chloride, 6.6 mM magnesium chloride, 0.7 mM dithiothreitol) containing 0.66 μg/μl histone H3 (Sigma-Aldrich Canada, Oakville, Ontario, Canada) for 2 h at 4°C. Reactions were started by introduction of 2 μCi of [32P]ATP (Amersham Pharmacia Biotech) and were carried out at 30°C for 30 min. Reactions were terminated by the addition of 2× sample loading buffer and boiling. Samples were resolved on 15% SDS-PAGE gels, which were then dried and subjected to analysis using a Molecular Dynamics phosphorImager (Sunnyvale, CA).
Under conditions of nitrogen starvation, diploid yeast of the Σ1278b background elongate, begin to bud in a unipolar manner, and form chains of cells in a process that has been referred to as pseudohyphal differentiation (Gimeno et al., 1992 ). Pseudohyphal differentiation is regulated by the RAS2 product via MAPK and cAMP/PKA signaling pathways (see INTRODUCTION). We reasoned that the C terminus of E1A might affect Ras2p function in yeast cells as it does ras function in mammalian cells, and examined whether expression of E1A in yeast affected diploid pseudohyphal growth. Expression of the C terminus (residues 187–289) of E1A fused to the yeast Gal4p DNA binding domain (DBD), the prokaryotic LexA DBD, or green fluorescent protein (GFP) (Figure (Figure1)1) strongly enhanced yeast pseudohyphal growth compared with yeast transformed with the parent vectors (Figure (Figure2).2). This effect appeared specific to E1A, as otherwise identical vectors expressing the comparably sized human papilloma virus (HPV) 16 E7 protein fused to the Gal4p or LexA DBD had no effect on pseudohyphal growth (Figure (Figure2). 2). Enhancement of pseudohyphal growth by E1A was observed on low-nitrogen medium but not on rich medium (our unpublished results). We also assessed the ability of a series of deletion mutants within the C-terminal domain of E1A to induce pseudohyphal growth (Figure (Figure3).3). The region of E1A required for induction of pseudohyphal growth was mapped to aa 284–289, the last five residues of E1A.
The MAP kinase signaling cascade consists of Ste20p, Ste11p, Ste7p, Kss1p, and the transcription factor Ste12p, which functions together with another transcription factor, Tec1p, to activate expression of genes required for pseudohyphal growth (Madhani and Fink, 1998 ). To test whether the C-terminal domain of E1A enhanced pseudohyphal differentiation through this MAPK cascade, we expressed the C-terminal region of E1A in diploid strains that are homozygous for the disruption of genes encoding components of this pathway. Disruption of STE7, KSS1, or STE12 abolished pseudohyphal growth in yeast that were not expressing E1A (Figure (Figure4).4). However, expression of the C-terminal region of E1A strongly induced pseudohyphal growth in these mutant strains (Figure (Figure4),4), indicating that this domain of E1A enhanced pseudohyphal growth independently of this yeast MAPK signal transduction pathway.
To test this conclusion further, we used an FG(TyA)::LacZ reporter construct, which is activated by Ste12p and Tec1p in response to MAPK activation under nitrogen starvation conditions (Madhani and Fink, 1997 ). In diploid wild-type yeast, this reporter was strongly stimulated by expression of the constitutively active STE11-4 allele. However, transcription from this reporter was not induced by expression of the C-terminal region of E1A (Table (Table3). 3). Moreover, expression of the C-terminal region of E1A did not induce reporter gene transcription in homozygous ste12Δ diploids (Table (Table3),3), although it enhanced pseudohyphal growth in this strain (Figure (Figure4C).4C). Thus, the biochemical analyses support the conclusion from the genetic studies that the C-terminal domain of E1A stimulates pseudohyphal growth independently of the MAPK cascade.
Ras2p also stimulates pseudohyphal growth via the cAMP/PKA pathway. In particular, Ras2p stimulates adenylate cyclase activity, and the resultant accumulation of cAMP activates Tpk2p, one of the three yeast isoforms of the catalytic subunit of PKA (Robertson and Fink, 1998 ). Tpk2p phosphorylates the transcription factor Flo8p, which is involved in the activation of transcription from genes required for pseudohyphal growth, including FLO11 (Pan and Heitman, 1999 ). To test if the C-terminal domain of E1A enhanced pseudohyphal differentiation through the cAMP/PKA signal transduction pathway, we first expressed the C-terminal region of E1A in diploid strains overexpressing genes that negatively regulate this pathway. To determine whether induction of pseudohyphal growth by the C-terminal region of E1A is dependent on increased levels of cAMP, we tested the effect of overexpressing PDE1 or PDE2, which encode low- and high-affinity phosphodiesterases, respectively (Ma et al., 1999 ). Although overexpression of either PDE1 or PDE2 inhibited pseudohyphal growth in yeast that were not expressing E1A, this had no effect on the ability of E1A to induce pseudohyphal growth (Figure (Figure5,5, A and B). Similarly, overexpression of BCY1, which encodes the negative regulatory subunit of PKA (Toda et al., 1987 ), abolished pseudohyphal growth in yeast that were not expressing E1A, but did not affect enhancement of pseudohyphal growth by the C-terminal domain of E1A (Figure (Figure5C).5C). Thus, pseudohyphal growth by E1A does not require increased levels of cAMP or enhancement of PKA signaling.
We further tested whether the C-terminal region of E1A functioned via the cAMP/PKA signal transduction pathway by disrupting components of this cascade and examining whether this would block the enhancement of pseudohyphal growth conferred by E1A. We expressed the C-terminal domain of E1A in diploid strains homozygous for disruptions in TPK2, FLO8, or FLO11. Each of these disruptions abolished pseudohyphal growth in yeast that were not expressing E1A, but had no effect on the ability of E1A to induce pseudohyphal growth (Figure (Figure5,5, D–F), strongly supporting the conclusion that the C-terminal domain of E1A enhances pseudohyphal growth independently of the cAMP/PKA signaling pathway.
PHD1 encodes a putative transcription factor that enhances pseudohyphal growth independently of the MAPK and cAMP pathways (Chandarlapaty and Errede, 1998 ; Pan and Heitman, 2000 ). Because the C-terminal region of E1A also functions independently of the MAPK and cAMP pathways, we asked whether it depends on Phd1p for function. Consistent with previous reports, the phd1Δ strain formed pseudohyphae poorly (Lo et al., 1997 ) and this was fully complemented by introduction of PHD1 (Figure (Figure6).6). Expression of the C-terminal domain of E1A failed to enhance pseudohyphal growth in this strain, indicating that it requires Phd1p for function.
To attempt to identify proteins with which the C-terminal domain of E1A interacts to stimulate pseudohyphal growth, we used the yeast two-hybrid screen. L40 yeast cells were transformed with a bait plasmid expressing the C-terminal region of E1A fused to the DNA binding domain of LexA and a library of yeast genomic DNA fragments fused to a transcriptional activation domain. We isolated one positive clone encoding a C-terminal fragment (aa 163–807) of the yeast dual specificity serine/threonine protein kinase Yak1p. Yak1p interacted specifically with the C-terminal domain of E1A and not with conserved region 2 of E1A or with comparably sized fragments of mouse CBP or human SUG1 fused to LexA (our unpublished results). Interestingly, Yak1p was originally identified as a negative regulator of cell growth that functions in opposition to the RAS-regulated cAMP/PKA pathway (Garrett and Broach, 1989 ; Ward and Garrett, 1994 ), but little is known about Yak1p function in yeast pseudohyphal signaling.
To identify the regions within the C-terminal domain of E1A that are required for interaction with Yak1p, two-hybrid interaction studies were performed with plasmids expressing a series of deletion mutants within the C-terminal region of E1A fused to the LexA binding domain. In addition to Yak1p, we used CtBP, the only other cellular protein known to interact with the C-terminal domain of E1A, as a control. The region of the C-terminal domain required for interaction with Drosophila CtBP was mapped to aa 271–284, which contain the CtBP binding motif (PLDLS), and aa 239–254 (Figure (Figure6).6). This is consistent with a previous report (Boyd et al., 1993 ), although the mutant Δ239–254 retained a measurable, but weaker interaction with human CtBP. This difference may be related to species differences between human and Drosophila CtBP or differences in the fusion constructs. Using the same deletion mutants, we determined that aa 187–221 and aa 239–284 of the C-terminal domain of E1A are required for interaction with Yak1p (Figure (Figure7). 7).
Yak1p-related proteins represent a novel subfamily of protein kinases with unique structural and enzymatic features, which have been categorized as the dual-specificity, Yak-related kinases (Dyrks) (Becker and Joost, 1999 ). The catalytic domain of yeast Yak1p shares the highest homology with the mammalian Dyrk1 proteins (Kentrup et al., 1996 ). To ask whether E1A might physically interact with the Dyrks, we conducted in vitro protein binding assays by using the rat DYRK1A and human DYRK1B products expressed as GST fusion proteins in E. coli (Figure (Figure8).8). Both the 243R and 289R E1A proteins were efficiently coprecipitated with the recombinant GST-Dyrk1A and GST-Dyrk1B, but not with GST alone, suggesting that mammalian Dyrk1A and Dyrk1B proteins physically interact with E1A.
An unusual enzymatic property of Yak1p-related kinases is their ability to catalyze tyrosine-directed autophosphorylation, as well as the phosphorylation of serine/threonine residues in exogenous substrates (Becker and Joost, 1999 ). To define the biochemical consequences of the interaction of E1A with mammalian Dyrks, we examined the effect of E1A on recombinant rat Dyrk1A kinase activity in vitro. Recombinant GST-E1A significantly enhanced the ability of rat GST-Dyrk1A to phosphorylate itself (Figure (Figure9A)9A) and histone H3 (Figure (Figure9B)9B) as substrate in a dose-responsive manner. No effect on Dyrk1A activity was observed upon addition of GST alone (our unpublished results). Thus, the interaction between Dyrk1A and E1A stimulated its kinase activity.
Overexpression of YAK1 in wild-type diploid yeast induced strong pseudohyphal growth compared with yeast transformed with a control vector (Figure (Figure10).10). However, a previous study had reported that YAK1 is not essential for pseudohyphal differentiation, because diploid yeast with homozygous deletions in YAK1 were still able to undergo pseudohyphal growth (Pan and Heitman, 1999 ). We also tested the ability of various regulators of pseudohyphal growth to induce filamentation in diploid yak1Δ yeast. Expression of the constitutively active RAS2VAL19, of components of the MAPK cascade (STE11–4, STE12, or TEC1), or of TPK2 (the central component of the cAMP/PKA pathway) all induced strong pseudohyphal growth in a wild-type strain, but not in the yak1Δ mutant (Figure (Figure11),11), indicating that Yak1p is essential for Ras2p regulation of pseudohyphal growth through both the MAPK and cAMP/PKA pathways. In contrast, overexpression of PHD1 induced strong pseudohyphal growth in both the wild-type and yak1Δ strain. Similarly, expression of the C terminus of E1A induced pseudohyphal growth in both the wild-type and yak1Δ mutant strains, although to a somewhat lesser extent in the yak1Δ mutant strain. These results are consistent with a role for Yak1p in modulating both the MAPK and cAMP/PKA signaling pathways that control pseudohyphal differentiation.
The E1A proteins of adenovirus have opposing effects on the functions of the mammalian ras oncogene product (Mymryk, 1996 ). Although E1A cooperates with activated ras to oncogenically transform cells (Ruley, 1983 ), it also suppresses ras-induced metastasis and tumorigenicity (Subramanian et al., 1989 ; Douglas et al., 1991 ; Linder et al., 1992 ; Boyd et al., 1993 ). Little is known about the mechanisms by which E1A modulates ras function in mammalian cells. The development of a simple model system in which the interactions between E1A and ras could be analyzed genetically would facilitate the elucidation of these interactions and their attendant regulatory pathways. Because extensive experimentation has shown that many regulatory mechanisms are conserved between the simple eukaryote S. cerevisiae and higher eukaryotic cells, we decided to investigate the effects of E1A on Ras2p function in this budding yeast.
In this study, we demonstrated that the C-terminal domain of adenovirus E1A strongly enhanced yeast pseudohyphal growth (Figure (Figure2)2) and this requires the last five residues of E1A (Figure (Figure3).3). E1A functions independently of the Ras2p-regulated MAPK and cAMP/PKA pathways to enhance pseudohyphal growth (Figures (Figures44 and and5),5), suggesting that it functions either downstream of these pathways or via a third parallel regulatory pathway. This is further supported by our observation that the induction of pseudohyphal growth by the C-terminal region of E1A requires Phd1p (Figure (Figure6),6), an enhancer of pseudohyphal growth that can function independently of the MAPK and cAMP/PKA pathways (Chandarlapaty and Errede, 1998 ; Pan and Heitman, 2000 ).
Using a yeast two-hybrid interaction screen to identify proteins that interact with the C terminus of E1A, we isolated a clone encoding aa 163–807 of the yeast Yak1p protein. Yak1p is a protein kinase of 807 amino acids that functions as a negative regulator of the Ras2p-regulated cAMP/PKA signal transduction pathway (Garrett and Broach, 1989 ). The cAMP/PKA signal transduction pathway is essential for the progression of yeast cells through the G0/G1 transition of the cell cycle (Garrett and Broach, 1989 ; Ward and Garrett, 1994 ). Yeast Yak1p was originally identified in a screen for mutants that suppress the growth defect in RAS mutant strains (Garrett and Broach, 1989 ). YAK1 mutation also restores growth in a strain lacking the three redundant PKA catalytic subunit genes (Ward and Garrett, 1994 ). Thus, Yak1p appears to act as an antagonist of the Ras2p-cAMP/PKA pathway and as a negative regulator of growth. However, Yak1p arrests growth only in yeast strains that are attenuated in the Ras2p-cAMP/PKA pathway and overexpression has no apparent effects on otherwise wild-type yeast cells (Garrett et al., 1991 ).
We performed a number of tests to determine whether Yak1p plays a role in regulating the signal transduction pathways that control pseudohyphal growth. Overexpression of YAK1 induced strong pseudohyphal growth in diploid yeast cells (Figure (Figure10).10). In addition, disruption of both copies of YAK1 in diploid yeast had profound effects on the ability of Ras2p, MAPK or cAMP/PKA components to stimulate pseudohyphal differentiation (Figure (Figure11).11). A role for Yak1p in pseudohyphal growth is consistent with previous work showing that Yak1p kinase activity is stimulated by nitrogen starvation (Garrett et al., 1991 ), the same signal used to stimulate pseudohyphal growth in these studies. Although our studies provide strong evidence that the Yak1p kinase modulates both the RAS-dependent MAPK and cAMP/PKA pathways, the exact effect on RAS-regulated signal transduction remains to be addressed. The recent observation that Yak1p interacts with the Hrt1p component of the Skp1p-Cdc53p-F-box complex (Uetz et al., 2000 ) may provide insight into the mechanism by which Yak1p affects pseudohyphal growth. The Skp1p-Cdc53p-F-box complex normally ubiquitinates the G1 cyclins Cln1p and Cln2p, signaling their proteolytic destruction (Skowyra et al., 1999 ). Interference with this process by Yak1p might stabilize Cln1p or Cln2p, both of which are essential for pseudohyphal growth, and each of which can promote cell elongation when overexpressed (Loeb et al., 1999 ). Alternatively, overexpression of Yak1p has also been shown to suppress the growth defects in late mitotic mutants, which characteristically exhibit increased levels of the G2/M cyclin Clb2p (Jaspersen et al., 1998 ). Interestingly, disruption of CLB2 induces constitutive pseudohyphal growth and overexpression of CLB2 can inhibit pseudohyphal growth (Ahn et al., 1999 ), suggesting that Yak1p could enhance pseudohyphal growth by reducing Clb2p expression or antagonizing Clb2p function.
Disruption of YAK1 had no effect on the ability of Phd1p and little effect on the ability of the C terminus of E1A to stimulate pseudohyphal growth (Figure (Figure11).11). These results are consistent with previous observations that Phd1p functions independently of the MAPK and cAMP/PKA pathways to induce pseudohyphal growth (Chandarlapaty and Errede, 1998 ). Importantly, although the C-terminal region of E1A binds to Yak1p, this appears to mediate only a small portion of the ability of E1A to induce pseudohyphal differentiation. This is consistent with our genetic (Figures (Figures44 and and5)5) and biochemical data (Table (Table3) 3) demonstrating that the C-terminal region of E1A induces pseudohyphal growth independently of the MAPK and cAMP/PKA pathways, but requires Phd1p (Figure (Figure6).6). This is also in agreement with our observation that the regions of E1A that interact with Yak1p are not essential for induction of pseudohyphal growth (Figures (Figures33 and and77).
Homologs of yeast YAK1 have been recently cloned and characterized. These include Drosophila MNB (Tejedor et al., 1995 ); Dictyostelium YAKA (Souza et al., 1998 ); and mammalian DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4, and DYRK4B (Kentrup et al., 1996 ; Becker et al., 1998 ). Although the precise function of the Dyrks has yet to be defined, they probably play an important role in regulating cell cycle and differentiation. Dictyostelium YAKA is required for the initiation of development, and overexpression of YAKA causes cell cycle arrest in nutrition-rich medium, promoting developmental events (Souza et al., 1998 ). In Drosophila, mutation of MNB results in specific defects in the development of the central nervous system (Tejedor et al., 1995 ). In humans, DRYK1A is located in the “Down syndrome critical region” of chromosome 21 (Chen and Antonarakis, 1997 ), suggesting that it too may be involved in development.
In addition to binding to Yak1p, we demonstrated that E1A interacts with rat Dyrk1A and human Dyrk1B in vitro (Figure (Figure8),8), suggesting that the C-terminal domain of E1A targets a conserved sequence present in both yeast Yak1p and mammalian homologs. Importantly, the interaction of E1A with rat Dyrk1A enhanced the ability of Dyrk1A to phosphorylate itself and histone H3 in vitro (Figure (Figure9),9), indicating that E1A could potentially activate Dyrk function at inappropriate times.
We determined that the interaction of E1A with Yak1p requires two separate regions spanning aa 187–221 and 241–284 of E1A (Figure (Figure7). 7). This region encompasses that required for the interaction of E1A with CtBP, but is more extensive. Mutants within the region spanning aa 241–284 are impaired for the ability to immortalize primary rodent cells, and fail to block ras-induced tumorigenesis and metastasis in rodent systems (Schaeper et al., 1995 ). Unfortunately, mutants in the region spanning aa 187–221 were not tested in that study. However, the existing data suggest a possible connection between these activities in mammalian cells and the interaction of E1A with Dyrks.
In conclusion, we have identified yeast Yak1p and the mammalian Dyrk1 proteins as a new family of cellular regulatory proteins targeted by the C-terminal region of E1A. Our data suggest that Yak1p modulates Ras2p signaling to regulate yeast pseudohyphal differentiation. By analogy, the Dyrk proteins may function similarly in mammalian cells to modulate ras function. Interestingly, the targeting of mammalian Dyrks by E1A may contribute to the ability of E1A to negatively modulate ras-induced tumorigenicity and metastasis.
We thank Dr. G. Hammond and Dr. J. Torchia for critical reading of the manuscript and Nik Avvakomov, Michael Shuen, and Jay Loftus for technical assistance. We thank Drs. G. Fink, J. Heitman, J. Thevelein, A. Dranginis, M. Christman, G. Chinnadurai, S. Parkhurst, D. Galloway, W. Becker, J. Torchia, and J. Broach for generous gifts of plasmids and strains. We also thank Dr. J. Pringle and anonymous reviewers for their helpful comments. This work was supported by grants from the Medical Research Council of Canada, The London Health Sciences Center, and The University of Western Ontario Academic Development Fund awarded to J.S.M., and National Institutes of Health Grant GM-28920 awarded to M.M.S. J.S.M. is supported by a Scholarship from the Canadian Institutes of Health Research.