Requirement of protein kinase C micro activation and calpain-mediated proteolysis for arachidonic acid-stimulated adhesion of MDA-MB-435 human mammary carcinoma cells to collagen type IV.
Arachidonic acid (AA) stimulation of adhesion of human metastatic breast carcinoma cells to collagen type IV depends on the protein kinase C (PKC) pathway(s) and is associated with the translocation of PKC mu from the cytoplasm to the membrane. In the present study, we have further explored the role of PKC mu in AA-stimulated adhesion. PKC mu activation site serines 738/742 and autophosphorylation site serine 910 are rapidly phosphorylated, and in vitro PKC mu kinase activity is enhanced in response to AA treatment. Inhibition of PKC mu activation blocks AA-stimulated adhesion. A phosphorylated, truncated species of PKC mu was detected in AA-treated cells. This 77-kDa protein contains the kinase domain but lacks a significant portion of the regulatory domains. Inhibition of calpain protease activity blocks generation of the truncated protein, promotes accumulation of the activated, full-length protein in the membrane, and blocks the AA-mediated increase in adhesion. p38 MAPK activity is also required for AA-stimulated adhesion. Activation of PKC mu and p38 are independent events. However, inhibition of p38 activity reduces calpain-mediated proteolysis of PKC mu and in vivo calpain activity, suggesting a role for p38 in regulation of calpain activity and a point for cross-talk between the PKC and MAPK pathways. These results support the hypothesis that AA stimulates activation of PKC mu, which is cleaved by calpain at the cell membrane. The resulting truncated kinase, as well as the full-length kinase, may be required for increased cell adhesion to collagen type IV. Additionally, these studies present the first evidence for calpain cleavage of a non-structural protein leading to the promotion of tumor cell adhesion.
Kennett
Sarah B
SB
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Science, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Roberts
John D
JD
Olden
Kenneth
K
D016718:Arachidonic Acid / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D024141:Collagen Type IV / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D024141:Collagen Type IV; D004791:Enzyme Inhibitors; D016718:Arachidonic Acid; C089212:protein kinase D; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D002154:Calpain