Type-2A protein phosphatase activity is required to maintain death receptor responsiveness.

Type-2A protein phosphatase (PP2A) is a key regulator in many different cell signaling pathways and an important determinant in tumorigenesis. One of the signaling targets of PP2A is the mitogen-activated protein kinase (MAPK/ERK) cascade. In this study, we wanted to determine whether PP2A could be involved in regulation of death receptor activity through its capacity to regulate MAPK/ERK. To this end, we studied the effects of two different routes of protein phosphatase inhibition on death receptor-mediated apoptosis. We demonstrated that the apoptosis mediated by Fas, TNF-alpha, and TRAIL in U937 cells is suppressed by calyculin A, an inhibitor of type-1 and type-2A protein phosphatases. The inhibition of the protein phosphatase activity was shown to subsequently increase the MAPK activity in these cells, and the level of activation corresponded to the degree of suppression of cytokine-mediated apoptosis. A more physiological inhibitor, the intracellular PP2A inhibitor protein I2(PP2A), protected transfected HeLa cells in a similar way from Fas-mediated apoptosis and induced activation of MAPK in I2(PP2A) transfected cells. A corresponding inhibition could also be obtained by stable transfection with a constitutively active form of the MAPK kinase, MKK1 (also referred to as MEK1). The inhibitor-mediated protection was highly efficient in preventing early stages of apoptosis, as no caspase-8 cleavage occurred in these cells. The observed apoptosis suppression is likely to facilitate the tumor-promoting effect of a range of different type-2A protein phosphatase inhibitors, and could explain the reported tumor association of I2(PP2A).

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR.

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1alpha-subunit at Ser293 (S293), which inactivates the complex. Because the alpha-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.

Characterization of a unique aspartate-rich protein of the SET/TAF-family in the human malaria parasite, Plasmodium falciparum, which inhibits protein phosphatase 2A.

A search for physiological inhibitors of protein phosphatases led to the identification of a Plasmodium falciparum (Pf) cDNA that had the potential to code for an aspartate-rich protein and hence named ARP. The PfARP was virtually identical to its Plasmodium berghei counterpart in gene structure and protein sequence. The PfARP coding sequence contained two introns, and the predicted protein contained 269 amino acid residues. Its primary structure showed significant similarity to eukaryotic proteins of the SET and TAF-family that included two inhibitors of mammalian serine/threonine protein phosphatase 2A (PP2A), namely I1(PP2A) and I2(PP2A). Like the SET and TAF proteins, it had an extremely acidic tail. The cDNA was confirmed by recombinant expression in bacteria. Native parasitic ARP was purified and was found to be highly thermostable. PfARP specifically inhibited the parasitic PP2A at nanomolar concentrations, with no effect on PP1, PP2B, PP5, or PPJ. Expression of PfARP in HeLa cells led to elevated phosphorylation of c-Jun, and activation of transcription factors AP1 and NF-kappa B. These functional properties are also characteristic of the SET/TAF-family proteins. The ARP mRNA and protein were detectable in all the erythrocytic asexual stages of the parasite, and the protein was located mainly in the parasitic cytoplasm. Thus, PfARP is a unique cytoplasmic member of the SET/TAF-family and a candidate physiological regulator of the Plasmodium PP2A.