Caracterização molecular da proteína DdI-2 e mapeamento de seus domínios de interação com a proteína fosfatase do tipo-1 de Dictyostelium discoideum / Molecular characterization of DdI-2 protein and domain mapping of Dictyostelium discoideum protein phosphatase type-1

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

The serine/threonine phosphatase of type-1 (PP1) is a ubiquous enzyme in the cells and tissues from several species studied and regulates numerous processes such as intermediate metabolism, mRNA splicing, transcription, and apoptosis. PP1 holoenzymes consist of a well-conserved catalytic subunit (PP1c) and one or more variable regulatory subunits. In mammals, more than fifty polypeptides that bind PP1c have been identified, originating holoenzymes with distinct cell locations and specificities. These proteins include cytosolic PP1c inhibitors such as inhibitor-1 (I-1), inhibitor-2 (I-2) and nuclear inhibitor of PP1 (NIPP-1). I-2 orthologs have also been described in Saccharomyces cerevisiae and Neurospora crassa. In the present work, we demonstrate that the genome of the social amoeba Dictyostelium discoideum has a single gene encoding for an I-2 ortholog (DdI-2). Northern blot analyses have shown that DdI-2 mRNA is expressed throughout Dictyostelium developmental cycle at variable levels. We also demonstrated that DdI-2 is a true PP1c inhibitor as its recombinant product is capable of inhibiting the phosphorylase phosphatase activity of wild-type PP1c (DdPP1c) and mutant (DdPP1cF269C) of D. discoideum and NcPP1c of N. crassa in vitro. DdPP1cF269C protein presents distint traits including higher stability, phosphorylase phosphatase activity and sensibility to calyculin A than the wild-type. These differences are originated from the replacement of a well conserved cisteine residue by a phenylalanine found in the wild-type. The wild-type and mutant DdPP1c have also been assayed in the presence of INc-1L and INc-1 which are orthologues to I-2 in N. crassa. Both purified recombinant proteins have shown inhibitory effects over phosphorylase phosphatase activities, with INc-1 being twice more potent than INc-1L. This might be due to the presence of an intron retention event in the latter that results in a insertion of 38 aminoacids. Our data also indicate that F269C mutation did not affect DdPP1c sensitivity to inhibition by all the three recombinant I-2 orthologues in vitro. Yeast two-hybrid assays using wild type (DdPP1c) and mutant (DdPP1cF269C) D. discoideum and N. crassa (NcPP1c) PP1c as preys and the putative inhibitor DdI-2 as a bait showed inequivocally that these proteins interacted in vivo. When the prey was INc-1 or INc-1L the interaction occured only with NcPP1c and was stronger with INc-1. The domains of DdI-2 involved in the interaction with DdPP1c were mapped by two-hybrid interaction assays with DdI-2 deleted mutants. These experiments have pointed out that the DdI-2 carboxi-terminus of ~100 aminoacids is not essential for the interaction but that the sum of all regions is responsible for the integrity of the interaction.

ASSUNTO(S)

dictyostelium discoideum ddl-2 protein protein phosphatase type-1 proteína fosfatase do tipo-1 dictyostelium discoideum proteína ddl-2

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