IGF-1 promotes β-amyloid production by a secretase-independent mechanism

β-Amyloid peptide (Aβ) is generated via the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Aβ. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of β-C-terminal fragment (β-CTF) and secreted Aβ. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of β-CTF-expressing cells with IGF-1 increased the levels of β-CTF and secreted Aβ. The IGF-1-induced augmentation of β-CTF was observed in the presence of γ-secretase inhibitors, but not in cells expressing β-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Aβ production through a secretase-independent mechanism involving APP phosphorylation.     

Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems

Amyloid-β peptide (Aβ), a putatively causative agent of Alzheimer’s disease (AD), is proteolytically derived from β-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease β-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Aβ production was modified by BACE1 inhibitors similarly to the production of endogenous murine Aβ in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP-APP by BACE1 or α-secretases. To render the assay specific for BACE1, the α-secretase cleavage site of SEAP-APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP-APP-EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.     

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Abstract

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.     

Enhanced generation of Alzheimer's amyloid-β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-β (Aβ) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCα down-regulation; (ii) PKCε up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity.     

Increased bisecting and core-fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on mutant human amyloid precursor proteins

Alteration of glycoprotein glycans often changes various properties of the glycoprotein. To understand the significance of N-glycosylation in the pathogenesis of early-onset familial Alzheimer’s disease (AD) and in β-amyloid (Aβ) production, we examined whether the mutations in the amyloid precursor protein (APP) gene found in familial AD affect the N-glycans on APP. We purified the secreted forms of wild-type and mutant human APPs (both the Swedish type and the London type) produced by transfected C17 cells and determined the N-glycan structures of these three recombinant APPs. Although the major N-glycan species of the three APPs were similar, both mutant APPs contained higher contents of bisecting N-acetylglucosamine and core-fucose residues as compared to wild-type APP. These results demonstrate that familial AD mutations in the polypeptide backbone of APP can affect processing of the attached N-glycans; however, whether these changes in N-glycosylation affect Aβ production remains to be established. Keiko Akasaka-Manya and Hiroshi Manya contributed equally to this work.     

Effect of Protein Kinase C Modulators on 14,15-Epoxyeicosatrienoic Acid Incorporation into Astroglial Phospholipids

Abstract: Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 ± 11,121 dpm/5 × 10⁶ cells) than incorporation of 8,9-EET (226,500 ± 5,567 dpm/5 × 10⁶ cells) and arachidonic acid (321,600 ± 1,200 dpm/5 × 10⁶ cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) ? phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4β-phorbol 12-myristate 13-acetate (4β-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4α-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4β-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid. Incorporation of 14,15-EET by PKC-down-regulated cells was inhibited by thimerosal, a known inhibitor of fatty acyl-CoA synthase. Our results suggest that the lower incorporation of 14,15-EET into astroglial cells may be due to modulation of PKC-mediated cellular mechanism(s).     

(−)-Epigallocatechin-3-Gallate Ameliorates Learning and Memory Deficits by Adjusting the Balance of TrkA/p75NTR Signaling in APP/PS1 Transgenic Mice

Alzheimer's disease (AD) is pathologically characterized by deposition of β-amyloid (Aβ) peptides, which closely correlates with the balance of nerve growth factor (NGF)-related TrkA/p75^NTR signaling. (?)-Epigallocatechin-3-gallate (EGCG) is used for prevention and treatment of many neurodegenerative diseases, including AD. However, whether the neuroprotective effects of EGCG treatment were via modulating the balance of TrkA/p75^NTR signaling was still unknown. In this study, we found that EGCG treatment (2 mg · kg ^–1 · day ^–1) dramatically ameliorated the cognitive impairments, reduced the overexpressions of Aβ(1–40) and amyloid precursor protein (APP), and inhibited the neuronal apoptosis in the APP/PS1 mice. Interestingly, the EGCG treatment enhanced the relative expression level of NGF by increasing the NGF/proNGF ratio in the APP/PS1 mice. Moreover, after EGCG treatment, TrkA signaling was activated by increasing the phosphorylation of TrkA following the increased phosphorylation of c-Raf, ERK1/2, and cAMP response element-binding protein (CREB), simultaneously the p75^NTR signaling was significantly inhibited by decreasing the p75^ICD expression, JNK2 phosphorylation, and cleaved-caspase 3 expression, so that the Aβ deposits and neuronal apoptosis in the hippocampus were inhibited.     

A secreted form of human ADAM9 has an alpha-secretase activity for APP

ADAM9 (MDC9, meltrin gamma) is a member of the ADAM family of metalloproteases, which play important roles in cell-cell fusion, intracellular signaling, and other cellular functions. Here we cloned a novel form of human ADAM9, designated hADAM9s (s for short), which lacks the carboxyl-terminus. Human ADAM9s was found to be secreted from transfected COS cells. RT-PCR analysis demonstrated that the mRNA for hADAM9s is expressed in human brain, liver, heart, kidney, lung, and trachea. When hADAM9s was co-expressed in COS cells with APP and treated with phorbol ester, the APP was digested exclusively at the alpha-secretory site. These results suggest that hADAM9s has an alpha-secretase-like activity for APP. Non-amyloidgenic cleavage of APP may occur at the plasma membrane. Our new results support a new therapeutic strategy to decrease in the Abeta content by directly activating ADAM9 in the extracellular space.     

Neuroprotective effects of blockers for T-type calcium channels

Background

Activation of the liver × receptors (LXRs) by exogenous ligands stimulates the degradation of β-amyloid 1–42 (Aβ42), a peptide that plays a central role in the pathogenesis of Alzheimer's disease (AD). The oxidized cholesterol products (oxysterols), 24-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), are endogenous activators of LXRs. However, the mechanisms by which these oxysterols may modulate Aβ42 levels are not well known.

Results

We determined the effect of 24-OHC and/or 27-OHC on Aβ generation in SH-SY5Y cells. We found that while 27-OHC increases levels of Aβ42, 24-OHC did not affect levels of this peptide. Increased Aβ42 levels with 27-OHC are associated with increased levels of β-amyloid precursor protein (APP) as well as β-secretase (BACE1), the enzyme that cleaves APP to yield Aβ. Unchanged Aβ42 levels with 24-OHC are associated with increased levels of sAPPα, suggesting that 24-OHC favors the processing of APP to the non-amyloidogenic pathway. Interestingly, 24-OHC, but not 27-OHC, increases levels of the ATP-binding cassette transporters, ABCA1 and ABCG1, which regulate cholesterol transport within and between cells.

Conclusion

These results suggest that cholesterol metabolites are linked to Aβ42 production. 24-OHC may favor the non-amyloidogenic pathway and 27-OHC may enhance production of Aβ42 by upregulating APP and BACE1. Regulation of 24-OHC: 27-OHC ratio could be an important strategy in controlling Aβ42 levels in AD.     

α-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid β (Aβ)-associated cell death

In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APP(LDN)) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.     

Macroautophagy as a Pathomechanism in Sporadic Inclusion Body Myositis

Skeletal muscle fibers show a high level of constitutive and starvation-induced macroautophagy. Sporadic Inclusion Body Myositis (sIBM) is the most common acquired skeletal muscle disease in patients above the age of 50 years and is characterized by inflammation and intracellular accumulation of aggregate-prone proteins such as amyloid precursor protein (APP)/β-amyloid, hyperphosporylated tau, and presenilin. In a recent study, we found that muscle fibers of sIBM patients show increased frequencies of Atg8/LC3⁺ autophagosomes and that intracellular APP/β-amyloid colocalized with Atg8/LC3 in degenerating fibers. Colocalization of APP/β-amyloid with LC3⁺ autophagosomes was further associated with upregulation of major histocompatibility complex (MHC) class I and class II molecules and T cell infiltration. These findings indicate that APP/β-amyloid is a substrate for autophagy in skeletal muscle fibers and suggest that degradation of aggregate-prone proteins via macroautophagy can be linked with both immune-mediated and degenerative tissue damage. A better understanding of this pathway in skeletal muscle and in the inflammatory environment of sIBM might provide a rationale for novel therapeutic strategies targeting pathogenic protein aggregation.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

Background: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.Results: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC βII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC α and βII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.Conclusions: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Influence of cholesterol and lovastatin on alpha-form of secreted amyloid precursor protein and expression of alpha7 nicotinic receptor on astrocytes

The influence of cholesterol and the lovastatin (cholesterol-lowering drug) on secretion of alpha-secretase cleavage product of amyloid precursor protein (APP) and expression of nicotinic acetylcholine receptors (nAChRs) was investigated in human HTB-15 astrocytes. The results showed that exposure of cholesterol to astrocytes inhibited the secretion of alpha-form of secreted APP (alphaAPPs) and reduced cell viability, while lovastatin enhanced the alpha-secretase processing on astrocytes; cholesterol treatment decreased expression of alpha7 nAChR, whereas lovastatin induced an up-regulation of the receptor; the increase in alphaAPPs resulted from lovastatin was partially inhibited by the alpha7 nAChR antagonists, alpha-bungarotoxin or methyllycaconitine; cholesterol or lovastatin did not influence either whole APP level or expression of alpha4 nAChR. We suggest that high dose of cholesterol may inhibit both the activity of alpha-secretase in APP metabolic processing and the expression of alpha7 nAChR, while lovastatin may stimulate alpha-secretase cleavage processing that might be regulated by alpha7 nAChR.     

Extracellular matrix regulates the amount of the β-amyloid precursor protein and its amyloidogenic fragments

We have studied the influence of the extracellular matrix (ECM) on the amount of β-amyloid precursor protein (APP) and C-terminal amyloid-bearing fragments in 3T3 fibroblasts. After incubation with ECM components, the cellular APP content of 3T3 cells changed. Besides, different substrata including collagen, fibronectin, laminin, vitronectin, and heparin, determined changes in the amount of a C-terminal 22 kDa-fragment. The regulation of amyloidogenic fragments by the ECM was transient; in fact, when 3T3 cells were plated on tissue culture dishes coated with collagen or vitronectin, maximal levels of the 22 kDa fragment were observed 12 h after plating; in the presence of fibronectin, the maximum level of the amyloidogenic fragment was obtained 36 h after plating. These results indicate that the ECM modulates in a transient way the generation of APP-derived polypeptides containing the amyloid-β-peptide (Aβ). The ECM does not have a generalized effect on 3T3 fibroblasts, because no significant differences in cell attachment, growth rate, whole-cell polypeptide pattern, β₁ integrin and α-tubulin levels were observed on cells grown on various matrix proteins. Laminin, collagen, and heparin also influence the level of an amyloidogenic fragment of 35 kDa in Neuro 2A neuronal cells, without a significant change in the neuronal marker acetylcholinesterase. In this case, however, a long-lasting response to ECM molecules was observed. These observations provide evidence that ECM molecules influence APP biogenesis, including the generation of amyloidogenic fragments containing the Aβ peptide. Our studies might prove significant to understand the localized increment of β-amyloid deposition in selected areas of the brain of Alzheimer's patients. © 1996 Wiley-Liss, Inc.     

Inhibitors of actin polymerization and calmodulin binding enhance protein kinase C-induced translocation of MARCKS in C6 glioma cells

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.