Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation

SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits

A new gene, SCH9, was isolated from Saccharomyces cerevisiae by its ability to complement a cdc25ts mutation. Sequence analysis indicates that it encodes a 90,000-dalton protein with a carboxy-terminal domain homologous to yeast and mammalian cAMP-dependent protein kinase catalytic subunits. In addition to suppressing loss of CDC25 function, multicopy plasmids containing SCH9 suppress the growth defects of strains lacking the RAS genes, the CYR1 gene, which encodes adenylyl cyclase, and the TPK genes, which encode the cAMP-dependent protein kinase catalytic subunits. Cells lacking SCH9 grow slowly and have a prolonged G1 phase of the cell cycle. This defect is suppressed by activation of the cAMP effector pathway. We propose that SCH9 encodes a protein kinase that is part of a growth control pathway which is at least partially redundant with the cAMP pathway

Two Phosphodiesterases from Ustilago Maydis Share Structural and Biochemical Properties with Non-Fungal Phosphodiesterases

The dependence of Protein Kinase A (PKA) activity on cAMP levels is an important facet of the dimorphic switch between budding and filamentous growth as well as for pathogenicity in some fungi. To better understand these processes in the pathogenic fungus Ustilago maydis, we characterized the structure and biochemical functions of two phosphodiesterase (PDE) genes. Phosphodiesterases are enzymes involved in cAMP turnover and thus, contribute to the regulation of the cAMP-PKA signaling pathway. Two predicted homologs of PDEs were identified in the genome of U. maydis and hypothesized to be involved in cAMP turnover, thus regulating activity of the PKA catalytic subunit. Both umpde1 and umpde2 genes contain domains associated with phosphodiesterase activity predicted by InterPro analysis. Biochemical characterization of recombinantly produced UmPde1 (U. maydis Phosphodiesterase I) and UmPde2 demonstrated that both enzymes have phosphodiesterase activity in vitro, yet neither was inhibited by the phosphodiesterase inhibitor IBMX. Moreover, UmPde1 is specific for cAMP, while UmPde2 has broader substrate specificity, utilizing cAMP and cGMP as substrates. In addition, UmPde2 was also found to have nucleotide phosphatase activity that was higher with GMP compared to AMP. These results demonstrate that UmPde1 is a bona fide phosphodiesterase, while UmPde2 has more general activity as a cyclic nucleotide phosphodiesterase and/or GMP/AMP phosphatase. Thus, UmPde1 and UmPde2 likely have important roles in cell morphology and development and share some characteristics with a variety of non-fungal phosphodiesterases

Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37-degrees-C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP

The regulation of the cAMP signalling pathway in the human pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is the causative agent of the disease Paracoccioidomycosis (PCM), which is one of the most prevalent systemic mycoses in Latin Amercia (Borges-Walmsley et al., 2002). P. brasiliensis is a thermally dimorphic fungus which undergoes morphological changes from a mycelial form at 26 C (environment) to a pathogenic yeast form at 37 C (human body) after inhalation of spores/conidia into the lungs of a human host (Nemecek et al., 2006). The cAMP pathway controls this morphological transformation in several fungi (Borges-Walmsley and Walmsley, 2000; Kronstad et al., 1998). G proteins are guanine-nucleotide (GDP or GTP) binding proteins that are generally associated with the cytoplasmic side of the plasma membrane. They receive signals from G protein-coupled receptors (GPCR). Adenylyl cyclase acts downstream of these G proteins. Ga subunits are required to regulate the activity of adenylyl cyclase (AC), which controls the level of cellular cAMP (Ivey and Hoffman, 2005). Protein Kinase A (PKA), which is activated by cAMP, is required for morphogenesis and virulence (Durrenberger et al., 1998; Sonnebom et al., 2000). The cAMP pathway in P. brasiliensis is poorly understood. However, recently the genes encoding a number of the components of the cAMP pathway have been cloned in our lab: these include the genes encoding three Ga proteins, Gpal-3, a Go protein, Gpb1; a Gy protein, Gpg1; Ras; adenylyl cyclase, Cyr1; and the catalytic subunit of PKA, Tpk2. Two-hybrid analyses confirmed that Gpa1 and Gpg1 interact with Gpb1. These data indicate the formation of a Gaβy trimer complex. A GST pull-down assay confirmed that Gpa1 and Gpb1 interacted with the N-teminus of adenylyl cyclase. Our hypothesis is that Gpa1 and Gpb1 modulate the activity of the AC/Tpk2 signalling pathway. Consistent with this hypothesis, we found changes in intracellular cAMP levels during the mycelium to yeast transformation that correlated with changing transcript levels of the signalling genes (Chen et al., 2007). We have established that Tpk2 interacts with the N-terminus of adenylyl cyclase, the G protein β subunit Gpb1 and with the co-repressor Tup1 by both two-hybrid and GST pull down analyses. This suggests that Tpk2 activity is required for feedback regulation of adenylyl cyclase to reduce cAMP levels. P. brasiliensis Tpk2-C-terminal 226-583-GFP and Tpk2 full length (FL) complemented the growth defect of a S. cerevisiae tpk2 temperature sensitive mutant strain SGY446 and induced the formation of pseudohyphue in the S. cerevisiae tpk2 mutant diploid strain XPY5a/a. Tpk2 C-tenninus has been over expressed in E. coli and in vitro PKA activity was measured. On the other hand we have also analysed the second catalytic subunit Tpk1, which failed to induce pseudohyphae in S. cerevisiae tpk1 inutant strain and is localised to the cytoplasm. Interestingly, the Pb Gβ subunit Gpb1 inhibited the development of pseudohyphae in TPK2 FL transformed yeast cells. Tpk2 C-terminus and Tpk2 FL co-transformed with Gpb-GFP were localized in the nucleus. Our hypothesis is that Gpb1 down regulates the activity of Tpk2, because Gpb1 binds to the catalytic C-terminal domain of Tpk2

Cell signalling in Paracoccidioides Brasiliensis

Paracocidioides brasiliensis (P. brasiliensis or Pb) is the etiological agent of paracocidioidomycosis which is the most prevalent systemic mycosis in South America. About 60% of the clinical cases are found concentrated in Brazil and the disease is a major threat for the public health there, p. brasiliensis is a dimorphic fungus because it undergoes a morphological switching from a mycelium to yeast form after shifting the temperature from 26 c to 37 c. Similar morphological changes have been implicated to be important in the pathogenicity of other dimorphic fungi and they appear to be under the control of the с AMP signalling transduction pathway. In order to establish the relationship of с AMP signalling pathway and the morphological change in p. brasiliensis, a project was initiated to clone the key components of the с AMP signalling pathway and analyze their functions. To this end, a genomic DNA library and two cDNA libraries oiPb were constructed. Degenerate primers were synthesized based on the sequence of genes of several other important fungi in GenBank. After amplification, specific PCR products were obtained and sequenced. The specific PCR products were used for labeling to screen libraries and their sequences were used for designing specific primers to do genomic walking and RACE-PCR. Both cDNA and genomic DNA sequences have been determined for the key components of the с AMP signalling pathway including adenylate cyclase (PbCYRl), three G protein α subunits (PbGPAl,PbGPA2 and PbGPA3), a G protein β subunit (PbGPBl), a G protein γ subunit (PbGPGl), a с AMP dependent protein kinase catalytic subunit (PbTPKJ), a с AMP depentdent protein kinase-like gene (PbTPKLl) and a TUP gene (PbTUPA).The interactions of the proteins encoded by the genes in the с AMP signaling pathway were studied with Clontech's Matchmaker yeast-two-hybrid System III. This approach demonstrated that the N-terminal third of adenylate cyclase PbCyrl 1 •678 can interact with PbGpal and PbGpbl. To further test if PbGpa2, PbGpa3, PbRasl, and PbActin can interact with adenylate cyclase, random mutagenesis libraries were made for these genes and screened with PbCyr1 1 •678, PbCyr1 600- 1316, PbCyri 1302 1876 and PbCyr1 1648-2100. It was demonstrated that PbCyrl 1-678, where the Ras association domain resides, can interact with truncated versions ofPbGpa2, PbGpa3, PbRasl, and PbActin, i.e., PbGpa21 -102, PbGpa21-183, PbGpa3 1-160, PbGpa3 1-213, PbRas1 1-83,PbRas 126-238, and PbActin 1-314. For the first time, the major components of Pb cAMP signaling pathway have been cloned and characterized; and we provide direct evidence that the G protein a subunits, G protein p subunit, RAS protein and actin interact directly with adenylate cyclase in fungal biology. This thesis funds the basis for the further study of the cAMP signalling pathway in P. brasiliensis. It may facilitate delineation of the mechanism for the dimorphic switching and the development of potentially novel antifungal drugs

Investigating the regulation of AMP-activated protein kinase and SNF1

AMP-activated protein kinase (AMPK) has long been known to play a critical role in the maintenance of energy homeostasis through direct interaction with or altering gene and protein expression of key players in diverse metabolic pathways. AMPK has been implicated in a number of diseases with roots in metabolic dysregulation, including obesity, type 2 diabetes and cancer. Elucidating the regulation of AMPK is an important part in understanding the progression of these diseases, and for developing small molecule modulators of AMPK activity which could have therapeutic applications. AMPK activity is determined by the phosphorylation status of T172 in the activation loop of the α subunit kinase domain. Binding of AMP to the γ subunit also increases its activity, primarily by preventing dephosphorylation of T172 but also by direct allosteric activation of the complex. The overall aim of this study was to investigate nucleotide regulation of AMPK. Site-directed mutagenesis studies showed that loss of highly conserved residues in γ1 disrupts regulation of both dephosphorylation and allosteric activation of AMPK by AMP. However, my studies revealed that these mutations do not have site-specific effects. The role of ADP in AMPK regulation was also investigated following the observation that this nucleotide also prevented dephosphorylation and inactivation of AMPK. The action of ADP on AMPK activity was characterised in wild-type complexes and insights from new structures of the active AMPK complex has provided insight into the molecular mechanism underlying AMP/ADP protection and dephosphorylation of T172. The yeast homologue of AMPK, SNF1, plays a central role in responding to glucose limitation and adaption to alternative carbon sources. Recombinant SNF1 complexes were used to show that ADP is the long-sought metabolite responsible for transmitting this low glucose signal and activates SNF1 by a similar mechanism to that seen in AMPK, preventing dephosphorylation and inactivation. Together these studies identify an important activator of both AMPK and SNF1, drawing parallels between these two systems and characterising a highly conserved regulatory mechanism, suggesting that ADP may represent a unifying trigger for activation of AMPK homologues in diverse species. Finally a potential link between AMPK and redox metabolism was identified in the form of NADH, opening new avenues of research in this field