ABSTRACT
Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.
Subject(s)
Acinar Cells/enzymology , Chloride-Bicarbonate Antiporters/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Salivary Glands/enzymology , Animals , CHO Cells , Chloride-Bicarbonate Antiporters/chemistry , Chloride-Bicarbonate Antiporters/genetics , Cricetulus , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Female , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Mutation , Phosphorylation , Protein Conformation , Salivary Glands/cytology , Structure-Activity RelationshipABSTRACT
The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.
Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Artificial Gene Fusion , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Gene Expression Profiling , Genes, Reporter , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Protein kinase A (PKA) is a broad specificity protein kinase that controls a physiological response following the increment of cAMP as a consequence of a particular stimulus. The specificity of cAMP-signal transduction is maintained by several levels of control acting all together. Herein we present the study of the regulation of the expression of each PKA subunit, analyzing the activity of their promoters. The promoter of each isoform of TPK and of BCY1 is differentially activated during the growth phase. A negative mechanism of isoform-dependent autoregulation directs TPKs and BCY1 gene expressions. TPK1 promoter activity is positively regulated during heat shock and saline stress. The kinase Rim15, but not the kinase Yak1, positively regulates TPK1 promoter. Msn2/4, Gis1, and Sok2 are transcription factors involved in the regulation of TPK1 expression during stress. TPK2, TPK3, and BCY1 promoters, unlike TPK1, are not activated under stress conditions, although all the promoters are activated under low or null protein kinase A activity. These results indicate that subunits share an inhibitory autoregulatory mechanism but have different mechanisms involved in response to heat shock or saline stress.
Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/biosynthesis , Cyclic AMP-Dependent Protein Kinases/biosynthesis , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae Proteins/biosynthesis , Transcription, Genetic , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Hot Temperature , Phosphorylation , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction/genetics , Stress, Physiological/genetics , Transcription Factors/geneticsABSTRACT
Previous studies on the dimorphic transition of Yarrowia lipolytica suggested opposite roles for MAPK and PKA pathways in this phenomenon. To obtain conclusive evidences for these opposite roles we isolated and disrupted the unique gene encoding the Pka catalytic subunit (TPK1). TPK1 was regulated only at the post-transcriptional level, with Pka activity increasing during yeast-like growth. tpk1 null mutants were viable and without growth defects, but more sensitive to different stress conditions. Deltatpk1 mutants were mating-deficient, and grew constitutively in the mycelial form, whereas Deltaste11 (Mapkkk-less)/Deltatpk1 double mutants grew in the yeast form, indicating that this is the default growth pattern of the fungus. Our data confirm that MAPK and PKA pathways operate in opposition during the dimorphic behavior of Y. lipolytica, but synergic in mating. These data stress the idea that in different fungi both signal transduction systems may operate distinctly or even be antagonistic or synergic in the coordination of cell responses to different stimuli.