title: CXCL12 and [N33A]CXCL12 in 5637 and HeLa cells: regulating HER1 phosphorylation via calmodulin/calcineurin.
abstract: In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.
authors.affiliation: Department of Medicine, Section of Hematology, University of Verona, Verona, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Rigo
authors.forename: Michele
authors.initials: M
authors.lastname: Gottardi
authors.forename: Ernesto
authors.initials: E
authors.lastname: Damiani
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bonifacio
authors.forename: Isacco
authors.initials: I
authors.lastname: Ferrarini
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Mauri
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Vinante
mesh_terms: D019943:Amino Acid Substitution; D019390:Arrestins / Q000235:genetics; D019703:Calcineurin / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D002147:Calmodulin; D054377:Chemokine CXCL12; D008024:Ligands; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C512478:EGFR protein, human; D066246:ErbB Receptors; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019703:Calcineurin