title: The effects of PMA and TFP and alterations in intracellular pH and calcium concentration on the membrane associations of phospholipid-binding proteins fodrin, protein kinase C and annexin II in cultured MDCK cells.
abstract: Annexin II, alpha-fodrin and protein kinase C (PKC) are associated with the cytoplasmic surface of the plasma membranes. When assayed with liposomes, they show affinity for acidic phospholipids and bind calcium ions. They also respond to or participate in cell signal transduction by altered membrane binding properties. In the present work we have studied the properties of these proteins in epithelial MDCK cells in response to elevated intracellular calcium ion concentration, lowered pH, treatment with tumor promoter phorbol myristoyl acetate (PMA) and calmodulin inhibitor trifluoperazine (TFP). In untreated polarized MDCK cells annexin II was seen both along the lateral walls and membranes of intracellular vesicles, fodrin was located along the lateral walls, whereas PKC was seen in the cytoplasm. There was no observable translocation of these proteins upon elevation of the intracellular calcium concentration using a calcium ionophore A23187. On the other hand, treatment with TFP led to a release of annexin II from the plasma membranes which was accompanied by a transient peak in the intracellular calcium. Treatment with PMA led to a loss of the cubic form of the cells, a slight elevation in the intracellular calcium concentration and a drop in the intracellular pH. Simultaneously fodrin was released from the lateral walls, but still remained insoluble in Triton X-100, PKC became associated with the intracellular membranes and fibers, whereas annexin II remained along the lateral walls. These changes could be prevented by clamping the intracellular pH neutral during PMA treatment. On the other hand, lowering of intracellular pH below 6.5 with the nigericin treatment led to a similar translocation of fodrin and PKC as PMA. This suggests that the protein redistribution is caused by cytoplasmic acidification and is due to an increased hydrophobicity and enhanced protonation of lipids and proteins. In contrast, no changes were seen in the annexin II distribution in response to altered pH. Hence, its release by TFP is presumably due to changes in the cationic properties of the inner phase of the plasma membrane. Thus, proteins which show similar binding properties with liposomes show different characteristics in their association with the intracellular membranes.
authors.affiliation: Biocenter, University of Oulu, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Vääräniemi
authors.forename: V
authors.initials: V
authors.lastname: Huotari
authors.forename: V P
authors.initials: VP
authors.lastname: Lehto
authors.forename: S
authors.initials: S
authors.lastname: Eskelinen
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014268:Trifluoperazine / Q000494:pharmacology*; D013049:Spectrin
chemical_list: D017306:Annexin A2; D002118:Calcium; D002147:Calmodulin; D002352:Carrier Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010743:Phospholipids; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D014268:Trifluoperazine; D013049:Spectrin