Identification of genes coding for serine/threonine phosphatases in Dictyostelium discoideum and functional characterization of type 4 protein phosphatase (PP4)

Author(s)

Leonardo Costa Fiorini

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2003-05-27

Examining board members

Aline Maria da Silva; Pio Colepicolo Neto; Marilis do Valle Marques; Ronaldo Bento Quaggio; Carlos Eduardo Winter

• Advisor: Aline Maria da Silva

Abstract

The serine/threonine protein phosphatases (PPs) are enzymes responsible for dephosphorylation of phosphoserine and/or phosphothreonine residues and are divided in two gene families designated as PPP and PPM. The PPP family is divided in five subfamilies, which comprise type 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) and 7 (PP7) phosphatases. This subdivision is based on aminoacid sequence similarity or enzyme domains. Novel PPs have been discovered in different organisms and classified in these families or subfamilies based on comparative sequence analysis. One of these is type 4 protein phosphatase (PP4), originally discovered in rabbit, which functions have been progressively uncovered. In this study our goal is to identify and characterize a novel serine/threonine phosphatase in Dictyostelium discoideum. We first screened a cDNA library and isolated a complete cDNA encoding the catalytic subunit of protein phosphatase 4 (PP4c), confirmed by manual DNA sequencing. The PP4c is an essential, single-copy gene located in chromosome 2, which encondes a mRNA constitutively expressed throughout D. discoideum life cycle. Immunodetection of PP4c performed with specific antibodies indicated corresponding protein levels. Overexpression of PP4c under a strong promoter caused no detectable phenotype. Subcellular localization of PP4c expressed as a GFP-fusion protein revealed its cytosolic location, in contrast to other organisms, where it has been reported to be enriched in centrosomes. We also describe here the genetic organization of PP4c, the genetic structure of all serine/threonine protein phosphatases belonging to the PPP family found in chromosome 2, and a phylogenetic analysis indicating relationships among these enzymes in D. discoideum and other selected organisms. (AU)