Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase.
Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin.
Shi
Heidi Y
HY
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Stafford
Lewis Joe
LJ
Liu
Zhisheng
Z
Liu
Mingyao
M
Zhang
Ming
M
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000473:pathology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D005260:Female; D017930:Genes, Reporter; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000970:Antineoplastic Agents; C107953:RAC1 protein, human; D011994:Recombinant Proteins; C085344:SERPIN-B5; D015843:Serpins; D018808:Transcription Factor AP-1; D008156:Luciferases; D005982:Glutathione Transferase; D048055:Mitogen-Activated Protein Kinase 8; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein