The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG

Abstract

Dictyostelium RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. We used a tetracycline-regulated protein expression system to study the effect of activated RasG, RasG(G12T), expression on the phosphorylation state of Dictyostelium proteins. Over 70 vegetative phosphoprotein components were resolved by two-dimensional (2-D) immunoblot analysis and of these 16 phosphothreonine and three phosphotyrosine protein components were found to reproducibly change upon RasG(G12T) expression. Thirteen of these were recovered from 2-D gels and identified by mass spectrometry of in-gel tryptic digestions. The proteins identified include the signaling proteins RasGEF-R and protein kinase B, the adhesion protein DdCAD-1, the cytoskeletal protein actin, the mitochondrial division protein FtsZA, and proteins involved in translation and metabolism. In addition to the direct demonstration of the phosphorylation of putative downstream targets of RasG activation, these findings reveal previously undetected phosphorylation of several proteins.

The Role of Tyrosine Phosphorylation in Regulation of Signal Transduction Pathways in Unicellular Eukaryotes

“…At the same time, phosphorylation of tyrosine-containing proteins of a molecular mass of 60 kDa rose significantly in the differentiation phase and remained elevated during the whole period of the fruiting body formation; this indicates an important role of various tyrosinecontaining D. discoideum proteins in the process of growth and differentiation. Recent investigations of effect of activated RasG on the phosphorylation state of D. discoideum proteins have shown that out of 70 vegetative phosphoprotein components resolved by two-dimensional (2-D) immunoblot analysis, three components were phosphotyrosine proteins (Secko et al, 2004). These data directly indicate participation of phosphotyrosine proteins in dictyostelium PTK-signaling.…”

An activated Ras protein alters cell adhesion by dephosphorylating Dictyostelium DdCAD-1

“…at 22 uC. The generation of the Ax2 : : MB, Ax2 : : MB-rasG(G12T), rasG null (IR17) and cadA null strains have been described previously (Secko et al, 2004;Tuxworth et al, 1997;Wong et al, 2002). Ax2 : : MB and Ax2 : : MB-rasG(G12T) transformants were maintained and grown in 10 mg G418 ml…”

“…In addition, RasG is activated in response to cAMP stimulation (Kae et al, 2004) and it influences the expression of the discoidin gene (Secko et al, 2001), a known molecular marker of the transition from growth to development (Zeng et al, 2000;Primpke et al, 2000). Recently, we reported the identification of proteins whose phosphorylation state was altered by the ectopic expression of RasG(G12T) (Secko et al, 2004). One of the phosphoproteins identified was DdCAD-1.…”

“…For RasG(G12T) induction, cells were centrifuged at 700 g for 4 min, washed three times in HL5 medium and then resuspended in HL5 medium containing no tetracycline (Secko et al, 2004). Strains were incubated in rotary-agitated suspension (175 r.p.m.)…”

“…Immunoblots were scanned using a UMAX-II scanner and densitometry was then performed using GeneQuant Analysis software (Molecular Dynamics). Two-dimensional (2D) electrophoresis and immunoblot analysis were performed as reported previously (Secko et al, 2004), except that 7 cm, pH 3-10, ready-to-use Immobiline DryStrips were rehydrated overnight in the presence of 100 mg total protein solution. Antibodies used were as follows: phosphothreonine polyclonal antibody (1 : 1000 dilution) (Cell Signalling Technology, cat.…”