Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (≥100 ng/ml) than in normal (C3H/OuJ) microglia (≤10 ng/ml). By contrast, TNFα production was not significantly increased in C3H/HeJ microglia even at 1 μg LPS/ml. Microglia expressed PKC isoforms α, β, δ, and ζ (but not γ and ε); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.

Signal transduction can involve the activation of protein kinase C (PKC) and the subsequent phosphorylation of protein substrates, including myristoylated alanine-rich C kinase substrate (MARCKS). Previously we showed that stimulation of phosphatidylcholine (PtdCho) synthesis by PMA in SK-N-MC human neuroblastoma cells required overexpression of MARCKS, whereas PKCalpha alone was insufficient. We have now investigated the role of MARCKS in PMA-stimulated PtdCho hydrolysis by phospholipase D (PLD). Overexpression of MARCKS enhanced PLD activity 1.3-2.5-fold compared with vector controls in unstimulated cells, and 3-4-fold in cells stimulated with 100 nM PMA. PMA-stimulated PLD activity was blocked by the PKC inhibitor bisindolylmaleimide. Activation of PLD by PMA was linear with time to 60 min, whereas stimulation of PtdCho synthesis by PMA in clones overexpressing MARCKS was observed after a 15 min time lag, suggesting that the hydrolysis of PtdCho by PLD preceded synthesis. The formation of phosphatidylbutanol by PLD was greatest when PtdCho was the predominantly labelled phospholipid, indicating that PtdCho was the preferred, but not the only, phospholipid substrate for PLD. Cells overexpressing MARCKS had 2-fold higher levels of PKCalpha than in vector control cells analysed by Western blot analysis; levels of PKCbeta and PLD were similar in all clones. The loss of both MARCKS and PKCalpha expression at higher subcultures of the clones was paralleled by the loss of stimulation of PLD activity and PtdCho synthesis by PMA. Our results show that MARCKS is an essential link in the PKC-mediated activation of PtdCho-specific PLD in these cells and that the stimulation of PtdCho synthesis by PMA is a secondary response.

MARCKS (myristoylated alanine-rich C-kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4β-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKCα to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKCα in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.

Abstract: To investigate the regulation of phorbol ester‐stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine‐rich protein kinase C substrate (MARCKS) and the α‐isoform of protein kinase C (PKC‐α) were overexpressed in a human neuroblastoma (SK‐N‐MC) cell line that does not increase PtdCho synthesis in response to 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl‐3H]choline was stimulated 1.88–2.34‐fold in the presence of 100–200 nM TPA. In clones overexpressing PKC‐α (30–40‐fold increased level of protein) or in mock‐transfected vector controls, TPA had much less of a stimulatory effect (1.04–1.43‐fold) on PtdCho synthesis. TPA caused translocation of PKC‐α and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK‐N‐MC cells, MARCKS is required for TPA‐stimulated synthesis of PtdCho, and PKC‐α alone is insufficient for supporting enhanced synthesis.

Microglia rapidly respond to Lipopolysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immune-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes, We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alanine-rich C kinase substrate) and MRP (MARCKS-related protein). Within 6 hours of addition, LPS induced a twofold increase in [H-3]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP, The differential effect of LPS on expression of MRP vs, MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS, TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription, CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS, Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1. (C) 1996 Wiley-Liss, Inc.

Neuroblastoma and glioma cells differentially express isoforms of protein kinase C (PKC) and myristoylated PKC substrates (e.g. MARCKS). Correlation with metabolism of membrane phospholipids suggests that PKC-α and MRCKS may be required to mediate phosphatidylcholine turnover stimulated by phorbol ester (β-TPA). To evaluate relationships to neural cell differentiation, SK-N-SH human neuroblastoma cells were treated with 20 nM β-TPA. In β-TPA-treated cells, growth arrest and differentiation occurred (neurite extension; 40–60% decrease in cell number, total protein and RNA). By day 4, mRNA for PKC-α and MARCKS increased and, after an initial decrease, PKC-α protein also increased. At day 4, phosphatidylcholine synthesis was 3–5 fold greater than in control cells. In contrast, C6 glioma cells treated with β-TPA showed no growth arrest, decreased PKC-α protein (<20%) and lower phosphatidylcholine synthesis. Thus, induced differentiation of human neuroblastoma cells involved increased expression of PKC-α and MARCKS and synthesis of phosphatidylcholine, consistent with involvement of PKC-α and MARCKS in regulation of phosphatidylcholine turnover during neurite growth.

Abstract: Expression of the protein kinase C substrate MARCKS and other heat‐stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB‐11 (SK‐N‐SH) neuroblastoma cells than in HTB‐10 (SK‐N‐MC) or mouse N1E‐115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H‐myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50‐kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E‐115 and HTB‐10) with less MARCKS, whereas neuromodulin (GAP‐43) was detected in N1E‐115 and HTB‐11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate‐labeled MARCKS and may be due to a conformational change affecting the C‐terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β‐12‐O‐tetradecanoylphorbol 13‐acetate resulted in three‐ to fourfold increased phosphorylation of MARCKS in HTB‐11 cells, with little increase noted in HTB‐10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin‐binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.