Interaction of serine/threonine protein phosphatase 5 with the protein products of tumour suppressor gene Tsc2

Abstract

Tuberous sclerosis (TSC) is a tumour disease caused by mutations in Tsc1 or Tsc2 genes. Both protein products of Tsc1 and Tsc2 form an intracellular complex possessing GTPase-activating (GAP) activity towards a small GTP binding protein Rheb. The activity of TSC1/2 complex is regulated by multiple phosphorylations of TSC2 mediated by several kinases, such as PKB/Akt, AMP-activated kinase (AMPK), ERK, MK2 and RSK1. So far, very little is known about the molecular mechanisms of TSC2 dephosphorylation. In the yeast two-hybrid screening, we have identified a number of potential TSC2 binding partners including protein phosphatase 5 (PP5). In this study, we provide the evidence that the interaction between TSC2 and PP5 also occurs in mammalian cells. Using TSC2 +/+ , p53-/mouse embryo fibroblasts (MEFs) transiently overexpressing myc-PP5, we showed in the immunoprecipitation assay that TSC2 specifically associates with myc-PP5 in exponentially growing cells. The physiological relevance of identified interaction, especially the involvement of PP5 in the dephosphorylation of major regulatory sites is currently under investigation.

PI3K/mTOR/S6K signaling pathway – new players and new functional links

“…However so far, very little is known about the molecular mechanisms of TSC2 dephosphorylation. In the yeast two-hybrid screening, we have identified a number of potential TSC2 binding partners including a protein phosphatase 5 (PP5) [51][52]. We provided the evidence that the interaction between TSC2 and PP5 also occurs in mammalian cells and found that this interaction is stronger in exponentially growing and serum stimulated cells when compared to serum starved cells.…”

Generation of monoclonal antibody against protein phosphatase 5

“…Recently we have identified PP5 as a new binding partner for a TSC2, a component of tuberous sclerosis complex TSC1/2 [28][29][30]. TSC1/2 complex serves as a point of integration between growth-stimulatory and growth-suppressive signaling upstream of mTOR in PI3Ê/Akt/mTOR signaling pathway.…”

Dephosphorylation of tuberous sclerosis complex 2 by serine/threonine protein phosphatase 5

“…Previously we have reported that serine/threonine protein phosphatase 5 (PP5) is a novel binding partner of TSC2 [13]. Furthermore, we demonstrated that PP5 interacts specifically with TSC2 not only in yeast two-hybrid system, but also in mammalian cells [14]. In order to establish functional link between TSC1/TSC2 and PP5 we studied the protein complex formation in starved cells and in response to serum stimulation.…”

“…Materials and Methods. Culturing and transfection of TSC2 +/+ p53 -/mouse embryonic fibroblasts (MEFs) was described previously in [14]. The production of anti-TSC2 mAbs (D6) is described in [15].…”

“…To analyze the interaction between TSC2 and PP5 in cells under different growth conditions, we have applied the immunoprecipitation assay followed by Western blotting. Initially TSC2 +/+ p53 -/-MEFs expressing TSC2 at high level [14], were transiently transfected with pcDNA3.1/Myc-PP5 construct. Western blot analysis of cell lysates with specific antibodies confirmed the expression of recombinant Myc-PP5 ( Fig.…”