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.

The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C delta.

Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-delta directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-delta can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-delta phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-delta does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-delta phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-delta.

Resistance to UV-induced apoptosis in Chinese-hamster ovary cells overexpressing phosphatidylserine synthases.

Externalization of PtdSer (phosphatidylserine) is an important event in signalling removal of apoptotic cells. In contrast with previous work [Yu, Byers, Ridgway, McMaster and Cook (2000) Biochim. Biophys. Acta 1487, 296-308] with U937 cells showing that specific stimulation of PtdSer biosynthesis during apoptosis was caspase dependent, PtdSer biosynthesis in CHO (Chinese-hamster ovary)-K1 increased 2.5-fold during UV-induced apoptosis but was not reversed by a caspase inhibitor, Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone). Also, in CHO-K1 cells, stimulation of synthesis was less specific for PtdSer as similar levels of stimulation were observed for sphingomyelin biosynthesis. Involvement of PtdSer synthase isoforms was tested in CHO-K1 cells overexpressing PSS I (PtdSer synthase I) and PSS II. Both types of transformed cells showed resistance to UV-induced apoptosis based on the decreased levels of caspase 3 activation and morphology changes; externalization of PtdSer was reduced with UV treatment even though expression of endogenous scramblase increased slightly. Serine-labelling experiments showed that PSS I- or PSS II-expressing cells had higher basal levels of PtdSer biosynthesis compared with vector control cells. When cells were exposed to UV light to induce apoptosis, PtdSer biosynthesis was further stimulated 1.5- and 2-fold in PSS I- and PSS II-expressing cells respectively compared with UV-treated vector cells. Caspase activation was not required, as Z-VAD-FMK did not change PtdSer synthesis. Although enhanced PtdSer synthesis was supposed to facilitate apoptosis, cells overexpressing PSS I and II were actually resistant to UV-induced apoptosis. Whereas enhanced PtdSer synthesis was associated with apoptosis, potential anti-apoptotic effects were observed when excess activity of these synthetic enzymes was present. This suggests a tightly regulated role for PtdSer synthesis and/or an important dependence on compartmentation of PSS enzymes in association with scramblase facilitated enrichment of this phospholipid at the cell surface.

The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.

Induction of protein kinase C substrates, Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), by amyloid beta-protein in mouse BV-2 microglial cells.

Microglial activation by amyloid beta-protein in senile plaques contributes to neurodegeneration in Alzheimer disease. In BV-2 microglial cells, amyloid beta-protein 1-40 (Abeta 1-40) elicited a dose-dependent increase (3-4 fold) of Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), two protein kinase C substrates implicated in membrane-cytoskeletal alterations underlying microglial adhesion, migration, secretion, and phagocytosis. Neither MARCKS nor MRP was induced by the amyloid fragment Abeta 25-35, although both Abeta 1-40 and Abeta 25-35 caused extensive aggregation of BV-2 cells. Interferon-gamma synergistically enhanced the induction by Abeta 1-40 of inducible nitric oxide synthase, but not MARCKS or MRP. Our results suggest that MARCKS and MRP may play important roles in microglia activated by amyloid peptides.

Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1.

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.