Role of cAMP-dependent protein kinase in controlling aggregation and postaggregative development in Dictyostelium.
We have examined the role of cAMP-dependent protein kinase (PKA) in controlling aggregation and postaggregative development in Dictyostelium. We previously showed that cells in which the gene encoding the PKA catalytic subunit has been disrupted (pkacat- cells) are unable to aggregate [S. K. O. Mann and R. A. Firtel (1991). A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium. Mech. Dev. 35, 89-102]. We show that pkacat- cells are unable to activate adenylyl cyclase in response to cAMP stimulation due to the inability to express the aggregation-stage, G-protein-stimulated adenylyl cyclase (ACA). Constitutive expression of ACA from an actin promoter results in a high level of Mn(2+)-stimulated adenylyl cyclase activity and restores chemoattractant- and GTP gamma S-stimulated adenylyl cyclase activity but not the ability to aggregate. Similarly, expression of the constitutively active, non-G protein-coupled adenylyl cyclase ACG in pkacat- cells also does not restore the ability to aggregate, although ACG can complement cells in which the ACA gene has been disrupted. These results indicate that pkacat- cells lack multiple, essential aggregation-stage functions. As the mound forms, high, continuous levels of extracellular cAMP functioning through the cAMP serpentine receptors activate a transcriptional cascade that leads to cell-type differentiation and morphogenesis. The first step is the induction and activation of the transcription factor GBF and downstream postaggregative genes, followed by the induction of prestalk- and prespore-specific genes. We show that pkacat- cells induce postaggregative gene expression in response to exogenous cAMP, but the level of induction of some of these genes, including GBF, is reduced. SP60 (a prespore-specific gene) is not induced and ecmA (a prestalk-specific gene) is induced to very low levels. Expressing GBF constitutively in pkacat- cells restores ecmA expression to a moderate level, but SP60 is not detectably induced. Overexpression of PKAcat from the Actin 15 (Act15), ecmA prestalk, and the PKAcat promoters in pkacat- cells result in significant aberrant spatial patterning of prestalk and prespore cells, as determined by lacZ reporter studies. Our studies identify new, essential regulatory roles for PKA in mediating multicellular development.
Mann
S K
SK
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Brown
J M
JM
Briscoe
C
C
Parent
C
C
Pitt
G
G
Devreotes
P N
PN
Firtel
R A
RA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology / Q000254:growth & development*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics; D051790:G-Box Binding Factors; D015971:Gene Expression Regulation, Enzymologic; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013431:Sulfates / Q000494:pharmacology; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051790:G-Box Binding Factors; D017895:Manganese Compounds; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D014157:Transcription Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; C039798:manganese sulfate