In vertebrates, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) regulates many cellular processes through its PtdIns(3,4,5)P3 lipid phosphatase activity, antagonizing PI3K (phosphoinositide 3-kinase) signalling. Given the important role of PI3Ks in the regulation of directed cell migration and the role of PTEN as an inhibitor of migration, it is somewhat surprising that data now indicate that PTEN is able to regulate cell migration independent of its lipid phosphatase activity. Here, we discuss the role of PTEN in the regulation of cell migration.
Intracellular signal transduction mediated by class I PI3K (phosphoinositide 3-kinase) enzymes regulates numerous diverse cellular processes, including cell proliferation, survival, growth and migration. Signalling mediated by these lipid kinases is characterized by their regulated activity, phosphorylating the relatively abundant membrane phospholipid PtdIns(4,5)P2, on the 3 position of the inositol ring, to produce the second messenger PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 in turn activates downstream responses via protein-binding targets that can selectively recognize this lipid and alter their behaviour, often through recruitment to particular membrane sites. One of the principal cellular antagonists of this signalling pathway is the tumour suppressor phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10), which dephosphorylates PtdIns(3,4,5)P3, converting it back into PtdIns(4,5)P2. Data supporting the role of PTEN as an inhibitor of PI3K signalling through its PtdIns(3,4,5)P3 phosphatase activity are overwhelming, and PTEN null cells display elevated rates of proliferation, growth and motility and resistance to apoptosis . However, when the effects of PTEN mutants on cell migration have been addressed, it has become clear that these effects are mediated independent of PI3K signalling, requiring a re-evaluation of the role of PTEN in the regulation of cell migration.
It has been clear for several years that PI3K signalling plays an important role in the regulation of cell migration and particularly in controlling the directionality of chemotaxis [2,3]. Studies in both mammalian cells and Dictyostelium amoebae show that many chemoattractants induce the localized accumulation of PtdIns(3,4,5)P3 at the leading edge of migrating cells. Given that the polarized localization of PtdIns(3,4,5)P3 occurs even in cells migrating up a very shallow chemoattractant gradient in which the activation of chemoattractant receptors varies with the ligand concentration, by as little as a few percentage from the front to the rear of the cell, this tight localization would appear to be a significant early event in the signalling pathways controlling directed cell migration. The functional significance of PI3K is supported by data showing that, in many cell types, cell migration in response to some growth factors and chemokines is blocked by relatively selective small molecule PI3K inhibitors [3,4]. Also, isoform-specific genetic deletion of murine PI3K isoforms shows that, in contrast with wild-type cells, neutrophils lacking PI3Kγ fail to produce PtdIns(3,4,5)P3 or induce chemotaxis in response to the chemoattractants fMLP (N-formylmethionyl-leucylphenylalanine) or C5a and, similarly, that mast cells lacking PI3Kδ fail to migrate in response to stem cell factor [3,5]. Finally, studies of Dictyostelium cells also show that cells lacking two of the class I PI3Ks fail to accumulate PtdIns(3,4,5)P3 at the leading edge and have greatly impaired chemotaxis towards cAMP or folate .
The proposed role for PI3K in the directional regulation of cell migration was also supported by extensive studies in Dictyostelium, which identified a role for the lipid phosphatase activity of Dictyostelium PTEN, opposing PI3K in this system [6,7]. Dictyostelium cells lacking PTEN often generate inappropriate pseudopodia at the sides and rear of migrating cells and are thus impaired in the speed and directionality of chemotaxis. Strikingly, analysis of the cellular localization of Dictyostelium PTEN showed that, during chemotaxis, the phosphatase was strongly localized to the membrane at the back of the cell, in contrast with its substrate PtdIns(3,4,5)P3, which is found concentrated at the leading edge. These results suggest a significant positive role for PTEN in the regulation of directed cell migration in this organism.
Regulation of mammalian cell migration by PTEN: not just PtdIns(3,4,5)P3
Several studies have indicated that the mechanism of action of mammalian PTEN in the regulation of cell migration is rather different from that in Dictyostelium. In mammalian cells of several lineages that genetically lack PTEN, these cells migrate faster than those expressing the phosphatase [8,9]. These studies have used both the re-expression of PTEN in human tumour cells that have lost the enzyme, and isogenic cells from gene-targeted mice. One detailed study using PTEN null Jurkat T-cells and PTEN expressing HL60 cells after PTEN knockdown by RNA interference found that PTEN expression affected the rate of both cell migration and stimulated actin polymerization, but not the directionality of this chemotaxis, in response to the chemoattractants CXCL12 or fMLP .
Significantly, strong evidence now indicates that the principal mechanism by which PTEN expression inhibits cell migration is independent of its PtdIns(3,4,5)P3 phosphatase activity and probably mediated largely through a currently unknown mechanism by the C2 domain of PTEN [11,12]. These studies, as well as our own work, have used a tumour-derived PTEN mutant, PTEN G129E (Gly129→Glu), which lacks significant lipid phosphatase activity, yet retains the protein phosphatase activity of the enzyme. These studies show that the C2 domain of PTEN, lacking any phosphatase activity, is sufficient to inhibit cell migration robustly. However, in the context of the full-length protein, the protein phosphatase activity of PTEN is required for the protein to inhibit cell migration, almost certainly autodephosphorylating the inhibitory C-terminal phosphorylation sites of PTEN. The autodephosphorylation of PTEN is supported by the finding that, while a phosphatase dead mutant PTEN does not inhibit cell migration, if a phosphatase dead mutation is combined with mutation of the C-terminal phopshorylation sites, this mutant protein regains the ability to inhibit migration. Direct evidence for autodephosphorylation of Thr383 of PTEN was also presented . However, these experiments do not exclude the significant possibility that the protein phosphatase activity of PTEN also acts on other non-PTEN substrates in order to inhibit migration.
Little is currently known regarding the mechanism of action of mammalian PTEN in the regulation of cell migration. Data using PTEN mutants lacking the C-terminal PDZ-binding site indicates that binding through a (currently unidentified) PDZ scaffold is required for PTEN to regulate cytoskeletal dynamics efficiently, such as membrane ruffling, cell spreading and cell migration [12,13]. Recent evidence from HL60 cells has supported the hypothesis that PTEN may also be transiently localized to the rear of mammalian cells during stimulated chemotaxis, probably through the localized activation of RhoA and ROCK (Rho-associated kinase), although aspects of this work are still controversial .
The finding that the expression of only the C2 domain of PTEN in PTEN null cells causes a potent inhibition of cell migration raises several possibilities regarding the function of several proteins related to PTEN. The human genome contains one processed pseudogene very closely related to PTEN and several genes encoding proteins, apparently lacking phosphatase activity, with significant sequence similarity to PTEN, including the three tensin family genes, as well as auxillin and cyclin G-associated kinase, each with approx. 30% amino acid identity through a region related to the PTEN phosphatase domain. More closely related to PTEN, however, is a group of at least seven gene-like sequences, each more closely related to each other than they are to PTEN, that contain the genes encoding two proteins, TPTE (transmembrane phosphatase with tensin homology) and TPIP (TPTE and PTEN homologous inositol lipid phosphatase), each with approx. 45% amino acid identity through the phosphatase domain when compared with PTEN and also encoding an adjoining C2 domain . It had been assumed that the other members of this gene family were likely to be pseudogenes, as they do not appear to contain open reading frames encoding potential phosphatase domain-containing proteins. However, since the naked C2 domain of PTEN has been shown to be capable of inhibiting cell migration, it raises the possibility that other members of this gene family encode inhibitors of cell migration.
Stem Cells and Development: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by T. Kouzarides (Cambridge, U.K.), S. Newbury (Newcastle upon Tyne, U.K.), B. Richardson (University College London, U.K.), R. Sablowski (John Innes Centre, Norwich, U.K.), D. Tosh (Bath, U.K.), M. Welham (Bath, U.K.) and A. Willis (Nottingham, U.K.).