Analysis of the YakA-regulated transcriptome and the role of KeaA in the regulation of Dictyostelium discoideum development

Author(s)

Luciana Mantzouranis

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2009-09-17

Examining board members

Glaucia Mendes Souza; Sergio Verjovski de Almeida; Suely Lopes Gomes; Marilis do Valle Marques; Lucile Maria Floeter Winter

• Advisor: Glaucia Mendes Souza

Abstract

The YakA is a protein kinase required for the regulation of several stress responses in Dictyostelium and is a key effector of the transition from growth to development in this microorganism. The gene keaA was isolated as suppressor of the yakA- mutant in a screen targeted to reveal genes involved in the survival to nitrosoative stress. The keaA gene codes a protein with six kelch domain repeats at the C-terminus, a zf-C3HC4 domain at the N-terminus, also called RING-finger, and a cysteine-rich sequence located in the mid-portion of the protein. The analysis of mutants deficient in keaA revealed a role for this gene also in the development process. keaA mRNA expression is induced when wild type cells grow and the food source becomes scarce. An induction of keaA mRNA expression also occurs during development. This induction is not observed in yakA- cells, indicating that YakA regulates KeaA. keaA deficient cells express low levels of pkaC, acaA and carA mRNA during aggregation in low cell densities, which may explain why the keaA deficient cells present a delay in development in low cell densities. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. The analysis of agreggation of keaA deficient cells during multicellular development indicated that KeaA is required for the cells to efficiently participate in the process. Cells over-expressing the cisteine-rich domain took the same time as the wild-type cells to reach the aggregation stage. However, these cells present smaller aggregates, culminants and fruiting bodies. Cells over-expressing the Kelch domain express high levels of acaA and carA after 8 hours of development, but the levels of pkaC are kept similar to those in wild-type cells. This could indicate that the Kelch domain induces the activation of PKA. However, this interaction was not observed when we conducted tests in a two-hybrid system. Additionally, gene expression in response to compounds that generate redox stresses was studied using cDNA microarrays. The results revealed a role of keaA in response to pre-starvation and control of cell cycle (AU)