Expression of MARCKS effector domain mutants alters phospholipase D activity and cytoskeletal morphology of SK-N-MC neuroblastoma cells.
Stable overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) is known to enhance phorbol ester stimulation of phospholipase D (PLD) activity and protein kinase Calpha (PKCalpha) levels in SK-N-MC neuroblastoma cells. In contrast, expression of MARCKS mutants (S152A or S156A) lacking key PKC phosphorylation sites within the central basic effector domain (ED) had no significant effect on PLD activity or PKCalpha levels relative to vector control cells. Like control cells, those expressing wild type MARCKS were elongated and possessed longitudinally oriented stress fibers, although these cells were more prone to detach from the substratum and undergo cell death upon phorbol ester treatment. However, cells expressing MARCKS ED mutants were irregularly shaped and stress fibers were either shorter or less abundant, and cell adhesion and viability were not affected. These results suggest that intact phosphorylation sites within the MARCKS ED are required for PLD activation and influence both membrane-cytoskeletal organization and cell viability.
Morash
Sherry C
SC
Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Room C-302 CRC, 5849 University Avenue, B3H 4H7, Halifax, NS, Canada.
Douglas
Donna
D
McMaster
Christopher R
CR
Cook
Harold W
HW
Byers
David M
DM
D000199:Actins / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009447:Neuroblastoma / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051571:Protein Kinase C-alpha; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1