Myristoylation-dependent and Electrostatic Interactions Exert Independent Effects on the Membrane Association of the Myristoylated Alanine-rich Protein Kinase C Substrate Protein in Intact Cells

Swierczynski, Sharon L.; Blackshear, Perry J.

doi:10.1074/jbc.271.38.23424

Cited by 67 publications

(49 citation statements)

References 50 publications

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“…A segment of DAKAP200 (residues 118 -148) includes a PSD-like cluster of 13 Lys, five Ser, and five large hydrophobic residues. Positive charge in PSD2S promotes electrostatic binding with negatively charged head groups of membrane phospholipids (53)(54)(55)(56). Intercalation of PSD hydrophobic side chains into the apolar interior of a bilayer further stabilizes association of PSD-containing proteins with membranes.…”

Section: Discussionmentioning

confidence: 99%

“…Intercalation of PSD hydrophobic side chains into the apolar interior of a bilayer further stabilizes association of PSD-containing proteins with membranes. N-terminal myristate and a PSD are critical features of MARCKS proteins, which mediate interactions between plasma membrane and F-actin (39,40,(51)(52)(53)(54)(55)(56). The MARCKS PSD is phosphorylated in situ by diacylglycerol-activated protein kinase C isoforms (39,40,51).…”

Section: Discussionmentioning

confidence: 99%

“…The MARCKS PSD is phosphorylated in situ by diacylglycerol-activated protein kinase C isoforms (39,40,51). Phosphorylation inhibits binding with membrane phospholipids, enables translocation of MARCKS from cell surface to cytoplasm, and promotes cytoskeleton remodeling (51)(52)(53)(54)(55)(56)(57)(58). The nonphosphorylated PSD sequesters calmodulin in a calcium-dependent manner (52,56,57).…”

Section: Discussionmentioning

confidence: 99%

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Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

Li¹,

Rossi²,

Hoheisel³

et al. 1999

Journal of Biological Chemistry

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A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI ϳ3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, ⌬DAKAP200. ⌬DAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. ⌬DAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and ⌬DAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; ⌬DAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.Cyclic AMP-dependent protein kinases (PKAs) 1 mediate actions of hormones that stimulate adenylate cyclase (1-4). Signals carried by cAMP often regulate activities of proteins that accumulate at discrete intracellular locations (e.g. ion channels in plasma membrane) (5-8). A kinase anchor proteins (AKAPs) guide transmission and routing of cAMP signals to such microenvironments. AKAPs have an avid binding site for regulatory subunits (RII) of PKAII isoforms 2 and distinct domains that target the tethered PKA holoenzyme to docking sites in organelles or cytoskeleton (5, 6, 9, 10). Accumulation of a high concentration of anchored PKAII in proximity with clustered substrate-effector protein promotes efficient reception, amplification, and precisely focused transmission of signals borne by cAMP (5-8).Several fundamental questions about AKAP⅐RII complexes remain unresolved. Current models suggest that AKAPs statically associate with docking sites in organelles. However, it is possible that AKAP⅐RII complexes shuttle dynamically between intracellular loci. This proposition has neither been excluded nor systematically investigated. Many physiological processes are coordinately regulated by groups of hormones. Homeostatic control is often achieved through synergistic or antagonistic interactions among signaling pathways that are activated by different second messenger molecules. A key question is how signals transmitted via AKAP⅐PKAII complexes are integrated with inputs from pathways controlled by other second messengers in order to regulate common effector proteins. PKA anchoring may be regulated by pretranslat...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

Li¹,

Rossi²,

Hoheisel³

et al. 1999

Journal of Biological Chemistry

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“…8) [13,21]. Proteins that use the myristate plus basic motif include pp60src [94,95], the HIV-1 viral protein Gag [96] and MARCKS [97]. Other Src family kinases and G α proteins use dual fatty acylation with palmitate and myristate to generate strong membrane binding [21].…”

Section: Myristoylated Proteinsmentioning

confidence: 99%

Protein myristoylation in health and disease

et al. 2009

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N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.Keywords Post-translational modification . Drug design . Myristoylation . Lipidation . Chemical proteomics Post-translational modification and myristoylationThe proteome is a larger and more dynamic entity than the genome, a result of both increased sequence diversity at the mRNA level due to alternative splicing of genes, and increased chemical and functional complexity at the protein level due to post-translational modification (PTM). This explains to some extent why larger genomes do not necessarily translate into more complex organisms. Posttranslational modification allows the incorporation of chemistries and new molecular functions that cannot be directly encoded by the gene sequence. Myristoylation is the attachment of a 14-carbon saturated fatty acid, myristate, to the N-terminal glycine of a subset of eukaryotic proteins [8,16,17]. Although often referred to as a PTM, it usually occurs co-translationally [18], when fewer than 100 residues have been polymerised by the

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“…This leads to the question of how MARCKS gets targeted to the lysosome. MARCKS is associated with the cellular plasma membrane predominantly by two mechanisms, the hydrophobic association of the aminoterminal myristate group with lipids of the membrane bilayer (16,44,45), and the electrostatic interaction of the highly basic phosphorylation site domain with acidic phospholipids of the membrane (16,(45)(46)(47)(48)(49). Following PKC-mediated phosphorylation of MARCKS, its affinity for the membrane is decreased and cytosolic MARCKS is increased (16, 45, 50 -52).…”

Section: Fig 3 Chromatographic Elution Profiles and Immunoblot Analmentioning

confidence: 99%

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH 4 Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 ؋ g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time-and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.The myristoylated alanine-rich C kinase substrate (MARCKS) 1 is a prominent cellular substrate for protein kinase C (PKC) (1, 2). Expression of this heat-stable, acidic protein is essential for life, as demonstrated by the perinatal death of mice that are completely deficient in MARCKS (3). Complete lack of expression leads to gross abnormalities of central nervous system development; however, heterozygous mice, which express MARCKS at 50% wild-type levels, appear to be normal.Cellular levels of MARCKS are regulated by both transcriptional and translational mechanisms (4 -14). In addition, we recently demonstrated that the cellular concentrations of MARCKS can also be regulated by a proteolytic event. This proteolytic cleavage results in amino-and carboxyl-terminal fragments of MARCKS that co-exist in cells with the full-length protein. This cleavage of MARCKS was inhibited in intact fi...

show abstract

Myristoylation-dependent and Electrostatic Interactions Exert Independent Effects on the Membrane Association of the Myristoylated Alanine-rich Protein Kinase C Substrate Protein in Intact Cells

Cited by 67 publications

References 50 publications

Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

Protein myristoylation in health and disease

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

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