Wnt-5a-induced phosphorylation of DARPP-32 inhibits breast cancer cell migration in a CREB-dependent manner.
Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 receptor or Galpha(s) expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr-34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Galpha(s)/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.
Hansen
Christian
C
Experimental Pathology, Department of Laboratory Medicine, Lund University, CRC, 205 02 Malmö, Sweden.
Howlin
Jillian
J
Tengholm
Anders
A
Dyachok
Oleg
O
Vogel
Wolfgang F
WF
Nairn
Angus C
AC
Greengard
Paul
P
Andersson
Tommy
T
D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology*; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002465:Cell Movement; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000378:metabolism*; D051157:Frizzled Receptors / Q000378:metabolism; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism*; D000071818:Wnt-5a Protein
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; C105574:FZD3 protein, human; D051157:Frizzled Receptors; D009687:Nuclear Proteins; C535048:PAGR1 protein, human; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D010769:Phosphothreonine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054645:Protein Phosphatase 1