Steroid hormone receptors, best known as transcriptional activators, are showing a new side to their character. A study by Dean Edwards and colleagues in Molecular Cell adds to growing evidence that steroid hormone receptors can modulate cytoplasmic signalling pathways by identifying a polyproline motif in the amino-terminal domain of the progesterone receptor (PR) that is able to activate Src kinases through a src-homology 3 (SH3) domain interaction.

The authors carried out a yeast two-hybrid screen using the amino terminus of PR as bait, and pulled out a clone corresponding to the carboxy-terminal-most SH3 domain of c-Cbl-associated protein (CAP). By expressing the SH3 domain of CAP and a panel of other signalling molecules as glutathione-S-transferase (GST) fusion proteins, the authors showed that PR bound directly — through SH3-domain interactions — to the Src family members, c-Src and Hck, as well as CAP. Conversely, they identified the extended left-handed polyproline (PPII) helix within the amino-terminal domain (PPPPLPPR) of PR as being necessary and sufficient for SH3-domain binding.

Of the other nuclear receptors tested — oestrogen, glucocorticoid, androgen and thyroid hormone — none were able to bind to SH3 domains in vitro. This is hardly surprising, as they all lack an obvious polyproline recognition sequence.

To test the hormone dependency of the PR–SH3 interaction in cells, Edwards et al. added R5020 — synthetic progestin — to normal breast epithelial cells expressing recombinant PR, and saw that it increased the interaction with GST–c-Src-SH3. R5020 also enhanced the interaction of PR and c-Src in a co-immunoprecipation assay in breast cancer cells that express both proteins endogenously.

So what effect does PR binding have on Src kinases? To investigate this, the authors expressed Hck with c-Src kinase (CSK) to phosphorylate tyrosine 527 of Hck, a residue involved in the autoinhibition of catalytic activity through intramolecular associations. Adding purified PR that was bound to R5020 stimulated Hck kinase activity in a concentration-dependent manner by displacing intramolecular interactions between the SH3 domain and a polyproline-like helix — the second important autoinhibitory mechanism.

The authors extended their study to show that progestin transiently stimulated c-Src activity in mammalian cells, which also led to a transient increase in the activity of mitogen-activated protein kinase (MAPK) — a downstream effector. Mutations in the second zinc finger of the DNA-binding domain of PR that destroy its transcriptional ability had no effect on the ability of progestin to stimulate c-Src activity. However, mutating three key prolines to alanines in the PPII motif of PR (PRmPro) abolished both the interaction with the SH3 domain of Src and Src's ability to be activated by progestin, supporting the idea that the transcriptional and cytoplasmic signalling functions of PR are totally independent.

So, what is the biological significance of PR–SH3 domain interactions? In normal mammary epithelium and breast cancer cells, progestin mainly exerts a growth inhibitory effect. In the present study, continuous treatment with R5020 for five days led to growth arrest in normal mammary cells expressing PR, whereas cells expressing PRmPro experienced a delayed onset of growth arrest. Similarly, expressing PRmPro in Xenopus oocytes had no effect on the rate of progesterone-induced maturation, in contrast to normal PR, which accelerated the rate of maturation.

So it seems that PR acts as a dual-function protein, first by directly interacting with target DNA in the nucleus and thereby modulating gene transcription, and second by interacting with SH3 domains to modulate cytoplasmic cell signalling pathways.