The tyrosine kinase Ron, receptor for MSP (macrophage-stimulating protein), displays several serine residues of unknown functions. Using [32P]H3PO4 metabolic labelling, we found that Ron is serine-phosphorylated and dephosphorylated in vitro by PP1 (protein phosphatase 1). PP1 associates with Ron obtained from cells of different origins. The association is stimulated by MSP or serum and is prevented by wortmannin, an inhibitor of the Akt/PKB (protein serine/threonine kinase B) pathway. Akt/PKB phosphorylates Ron Ser-1394, thus providing a docking site for 14-3-3 (scaffold proteins binding to phosphoserine/phosphothreonine-containing sequences). In living cells, PP1 binds to the Ron mutant S1394A, but the association is no longer regulated by serum, MSP or wortmannin. The role of PP1 association with Ron is highlighted by (1) Ser-1394 dephosphorylation by PP1 in vitro and in living cells, (2) loss of 14-3-3 association with Ron after Ser-1394 dephosphorylation by PP1 in vitro and (3) an increase in 14-3-3 association after PP1 inactivation in living cells. These results suggest that PP1 can modulate the downstream Ron signalling generated by MSP via Akt/PKB and 14-3-3 binding. This is the first report on ligand-regulated association of PP1 with a growth factor receptor.
- Akt/protein serine/threonine kinase B (PKB)
- macrophage-stimulating protein (MSP)
- protein phosphatase
- protein phosphorylation
- receptor tyrosine kinase
Abbreviations used: Akt/PKB, protein serine/threonine kinase B; FCS, foetal calf serum; HGF, hepatocyte growth factor; LB broth, Luria–Bertani broth; PP2A, protein phosphatase 2A; PTP, protein tyrosine phosphatase; RTK, receptor tyrosine kinase; MSP, macrophage-stimulating protein; GST, glutathione S-transferase.
- The Biochemical Society, London ©2003