Multiple roles of the novel protein tyrosine phosphatase PTP3 during Dictyostelium growth and development

Abstract

PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic protein tyrosine phosphatase domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow-growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys to Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of various sizes. Antiphosphotyrosine Western blot (immunoblot) analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with nonnutrient buffer led to reciprocal changes in the pattern of several phosphotyrosine proteins, including a protein of approximately 130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested that p130 is a potential substrate of PTP3. A transient posttranslational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data obtained strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2.