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Role of 14-3-3β, Raf-1 & MAPK

Introduction

Chlamydiae have to interact with the host cell across the surrounding inclusion membrane in order to ensure their survival. The Inc series of inclusion membrane located proteins is likely to be one of the ways they do this [See: inclusion proteins].  IncG is co-transcribed with IncD, E & F within 2 hours after infection [Scidmore & Hackstadt, 2001]. IncA and IncG are phosphorylated, and their C terminal domains are exposed to the host cell cytoplasm where they are capable of interacting with host cell signalling systems [Rockey et al., 1995, 1997, 2002;  Scidmore-Carlson, 1999; Scidmore & Hackstadt, 2001]. Using yeast two hybrid technology [lay reader: a technique in molecular biology which enables one to identify protein protein interactions]  a host cell protein, 14-3-3beta, that binds to phosphoserine at a conserved motif (RS164RS166F) on C. trachomatis IncG has been identified [Scidmore & Hackstadt, 2001]. [NB. This association is specific to the IncG of Chlamydia trachomatis and the closely related C. muridarum but not to Chlamydophila species including  C. pneumoniae and C. psittaci (see presentation Fig 5 below). If confirmed with other species, this might be a possible basis for some of the observed differences between  Chlamydia and Chlamydophila spp].

14-3-3 is a dimeric, cytosolic, conserved host cell signalling protein. Several distinct isoforms of the protein occur (Fig 6 below) and there are more than 100 known ligands which bind to it, including IncG. The protein is implicated in a wide variety of signal transduction pathways, including apoptosis, cell cycle control and mitogenic signal transduction [Fu et al., 2000; Scidmore, 2002]. Thus 14-3-3 interactions might be a mechanism by which a wide variety of cell processes might be modulated. This is further suggested by the observation that, in infected cells, there is both the induction and disappearance of 14-3-3 binding sites on several proteins. Accordingly, Marci Scidmore investigated the interaction of 14-3-3 with Raf-1, a 14-3-3 ligand that is involved in mitogen activated protein kinase (MAPK) signal transduction. In this presentation, reproduced here by kind permission of  Marci Scidmore and given  to the 10th International Chlamydia Conference in June 2002, she shows that C. trachomatis L2 infection leads to decreases in Raf-1 and alterations in MAPK signalling pathways. For a different interaction of chlamydiae with host cell signalling see Coombes & Mahony, 2002.

[MEW November 2002]

Marci Scidmore's presentation:

Fig 1. Title of presentation. This presentation © Marci Scidmore 2002.	Fig 2. C. trachomatis IncG properties.	Fig 3.  Association of 14-3-3 with the C. trachomatis inclusion. See Scidmore & Hackstadt, 2001.	Fig 4. Diagram of the interaction of IncG in the inclusion membrane with 14-3-3 in the host cell cytoplasm.
Fig 5. 14-3-3β association with the chlamydial inclusion is specific for Chlamydia spp but not Chlamydophila spp.	Fig 6. There are 7 mammalian isoforms of 14-3-3, with isoforms beta and tau or theta showing the strongest localisation to the inclusion membrane.	Fig 7. Properties of 14-3-3.	Fig 8. The possible effects of the recruitment of 14-3-3 to the chlamydial inclusion membrane.
Fig 9. An immunoblotting study through the chlamydial growth cycle using an antiserum specific for the 14-3-3 phosphoserine binding motif. The arrows show the position of new 14-3-3 binding sites that appear in infected cells 24 and 36 hours post infection. The asterisks denote binding sites that are absent in infected cells 24 hours post infection. CAM is a chloramphenicol control where 80S host cell protein synthesis in inhibited.	Fig 10. Diagram of the possible interactions of 14-3-3beta with Raf-1, a ligand involved in MAPK signal transduction via MEK and ERK.	Fig 11. Coprecipitation of cMyc tagged 14-3-3beta and endogenous Raf-1  from HeLa cells with antisera specific for cMyc  or Raf-1. In uninfected cells (UI) there is a strong association of 14-3-3beta with Raf-1. In contrast, in L2-infected cells there is a marked decrease in association of Raf-1 with 14-3-3β.	Fig 12. Decrease in the steady state levels of Raf-1 through the C. trachomatis L2 growth cycle.
Fig 13.ERK1/2p, p38p and JNKp are components of the MAPK signalling cascade that are transiently induced during infection. Only the activation of ERK1/2p is sustained over the entire C. trachomatis developmental cycle.	Fig 14. Conclusions.	Fig 15. Collaborators in the study.

Coombes, B.K. & Mahony, J. B. (2002). Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells. Cellular Microbiology 4, 447 - 460.

Fu, H., Subramanian, R. R. & Masters, S. C. (2000). 14-3-3 proteins: structure, function, and regulation. Annual Reviews of Pharmacology and Toxicology 40, 617 - 647. Full article

Rockey, D. D., Heinzen, R. A. & Hackstadt, T. (1995). Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Molecular Microbiology 15, 617 - 626. Rockey, D. D., Grosenbach, D., Hruby, D. E. et al., (1997). Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion. Molecular Microbiology 24, 217 - 228.

Scidmore, M. A. & Hackstadt, T. (2001). Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG. Molecular Microbiology 39, 1638 - 1650.

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