Deborah L. Bennett

Redox regulation of PI 3‐kinase signalling via inactivation of PTEN

The tumour suppressor PTEN is a PtdIns(3,4,5)P3 phosphatase that regulates many cellular processes through direct antagonism of PI 3‐kinase signalling. Here we show that oxidative stress activates PI 3‐kinase‐dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P3 levels and activation of the downstream PtdIns(3,4,5)P3 target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant‐dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P3 and oxidants are produced concurrently.

Targeting mutants of PTEN reveal distinct subsets of tumour suppressor functions

The tumour suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase which can antagonize the phosphoinositide 3-kinase (PI 3-kinase) signalling pathway, promoting apoptosis and inhibiting cell-cycle progression and cell motility. We show that very little cellular PTEN is associated with the plasma membrane, but that artificial membrane-targeting of PTEN enhances its inhibition of signalling to protein kinase B (PKB). Evidence for potential targeting of PTEN to the membrane through PDZ domain-mediated protein-protein interactions led us to use a PTEN enzyme with a deletion of the C-terminal PDZ-binding sequence, that retains full phosphatase activity against soluble substrates, and to analyse the efficiency of this mutant in different cellular assays. The extreme C-terminal PDZ-binding sequence was dispensable for the efficient down-regulation of cellular PtdIns(3,4,5)P3 levels and a number of PI 3-kinase-dependent signalling activities, including PKB and p70S6K. However, the PDZ-binding sequence was required for the efficient inhibition of cell spreading. The data show that a PTEN mutation, similar to those found in some tumours, affects some functions of the protein but not others, and implicate the deregulation of PTEN-dependent processes other than PKB activation in the development of some tumours. Significantly, this hypothesis is supported by data showing low levels of PKB phosphorylation in a glioblastoma sample carrying a mutation in the extreme C-terminus of PTEN compared with tumours carrying phosphatase-inactivating mutations of the enzyme. Our data show that deregulation of PKB is not a universal feature of tumours carrying PTEN mutations and implicate other processes that may be deregulated in these tumours.

Proprotein-processing endopeptidases of the insulin secretory granule

Enzymological studies have implicated two Ca2+ dependent endopeptidases in the conversion of proinsulin to insulin: a type 1 activity and a type 2 activity which cleave on the C-terminal side of R31R32 and K64R65 in proinsulin, respectively. These activities were further characterized and their relationship to the mammalian family of subtilisin-like proteases was investigated. PC2 was expressed in neuroendocrine tissues and in insulinoma secretory granule fractions predominantly as a 65kDa protein. On anion-exchange chromatography of solubilized granules, PC1/3 immunoreactivity comigrated with a peak of type 1 activity whereas PC2 immunoreactivity coeluted with the peak of type 2 endopeptidase activity. PC2 antiserum gave a specific immunoprecipitation of type 2 activity from insulin granule extracts. It was concluded that the PC2 gene-product has type 2 endopeptidase activity.

Proinsulin-Processing Endopeptidases

Knowledge of the gene structure of secreted proteins and analysis of their biosynthesis has revealed that many are initially produced as larger biologically inactive precursors that are subjected to limited proteolysis at sites marked by clusters of basic amino acids in characteristic linear sequences. Peptide hormones and neurotransmitters that are secreted via the regulated secretory pathway in neuroendocrine tissues are generally processed at pairs of basic amino acids, typically Lys Arg and Arg Arg and much less frequently at Lys Lys or Arg Lys sites. Growth factors and receptor molecules that follow the constitutive pathway are processed at sites marked by a more complex array of basic amino acids, typically a pair of basic amino acids with a further basic amino acid in the P4 position (for review, see ref. 7). In the majority of cases where the most C-terminal amino acid in the cluster is an arginine residue, cleavage of the amide bond occurs on the C-terminal side of this arginine. When it is a lysine residue, cleavage often occurs on the N-terminal side and probably involves a different enzymatic process in many cases. Basic amino acids exposed at the C-termini after endoproteolytic cleavage are removed by carboxypeptidase H (E) (CPH) within the regulated pathway of secretion. Whether a similar activity is present in the constitutive pathway is not clear.