Evidence for a role of MRCK in mediating HeLa cell elongation induced by the C1 domain ligand HMI-1a3.
Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of β2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology.
Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland. Electronic address: virpi.talman@helsinki.fi.
Virpi
V
Talman
Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland.
Gergana
G
Gateva
Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland.
Marja
M
Ahti
Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland.
Elina
E
Ekokoski
Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland.
Pekka
P
Lappalainen
Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland.
Raimo K
RK
Tuominen
D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects*; D051338:Cofilin 1 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022001:Focal Adhesions / Q000187:drug effects / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D065636:Myotonin-Protein Kinase; D010641:Phenotype; D010766:Phosphorylation; D010795:Phthalic Acids / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D022502:Stress Fibers / Q000187:drug effects / Q000201:enzymology; D013997:Time Factors; D014162:Transfection
C495810:CFL1 protein, human; D051338:Cofilin 1; D010795:Phthalic Acids; D047428:Protein Kinase Inhibitors; C000592883:bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate; C105518:CDC42BPA protein, human; C089212:protein kinase D; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C
C1 domain; CID755673 (PubChem CID: 755673); Cell elongation; Cell proliferation; Chelerythrine (PubChem CID: 72311); Gö6983 (PubChem CID: 3499); Isophthalate; Myotonic dystrophy kinase-related Cdc42-binding kinase; Phorbol 12,13-dibutyrate (PubChem CID: 37783); Phorbol 12-myristate 13-acetate (PubChem CID: CID 27924); Protein kinase C; U0126 (PubChem CID: 3006531)