cGMP-dependent phosphorylation and degradation of myristoylated alanine-rich C-kinase substrate

In the mammalian brain, nitric oxide (NO) has been implicated in neuronal signal transmissions. NO stimulates guanylate cyclase to increase intracellular cGMP, which in turn activates cGMP-dependent protein kinases (PKG), but the targets of PKG in the brain have not fully been understood. In this study, we examined cGMP-dependent phosphorylation of proteins in rat brain and found that one of the possible substrates was myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding membrane-associated protein that regulates cell adhesion. In addition, possible degradation products of MARCKS were observed after transfection of PKG or stimulation with 8pCPT-cGMP. Western blot analysis showed that the MARCKS protein levels were decreased when the cells were stimulated with 8pCPT-cGMP. These results suggest that MARCKS is a target of PKG, and PKG-dependent phosphorylation of MARCKS results in its degradation to reduce its protein levels in the cells.     

Overexpression of the myristoylated alanine-rich C-kinase substrate inhibits cell adhesion to extracellular matrix components.

Mice lacking the myristoylated alanine-rich C-kinase substrate, or MARCKS protein, exhibit abnormalities consistent with a defect in the ability of neurons to migrate appropriately during forebrain development. To investigate the possibility that this phenotype could be due to disruption of normal cellular adhesion to extracellular matrix, an assay was developed in which 293 cells co-expressing MARCKS and green fluorescent protein were tested for their adhesion competence on various substrates. Fluorescence-activated cell sorting of adherent and non-adherent green fluorescent protein-expressing cells demonstrated that wild-type MARCKS inhibited adhesion of cells to fibronectin, whereas a non-myristoylated mutant did not inhibit adhesion of cells to a variety of substrates. The fibronectin competitive inhibitor RGD peptide inhibited adhesion of cells expressing all MARCKS variants equally. Cytochalasin D inhibited the adhesion of cells expressing non-myristoylated MARCKS, but did not further decrease the adhesion of cells expressing adhesion-inhibitory proteins. Confocal microscopy demonstrated the presence of inhibitory, myristoylated MARCKS at the plasma membrane, suggesting that localization at this region might be important for MARCKS to inhibit cellular adhesion. These data suggest a possible myristoylation-dependent function of MARCKS to inhibit cellular adhesion to extracellular matrix proteins, indicating a potential mechanism for the cell migration defects seen in the MARCKS-deficient mice.     

Location of the myristoylated alanine-rich C-kinase substrate (MARCKS) effector domain in negatively charged phospholipid bicelles

The effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS-ED) is a highly basic, unstructured protein segment that is responsible for attaching MARCKS reversibly to the membrane interface. When attached to the interface, it also has the capacity to sequester phosphoinosities, such as PI(4,5)P(2), within the plane of the bilayer. Here, the position of the MARCKS-ED was determined when bound to phospholipid bicelles using high-resolution NMR methods. Two sets of data indicate that the phenylalanine residues of the MARCKS-ED are positioned within the membrane hydrocarbon a few angstroms from the aqueous-hydrocarbon interface. First, short-range nuclear Overhauser effects are detected between the aromatic side chains and the lipid acyl chain methylenes. Second, paramagnetic enhancements of nuclear relaxation, produced by molecular oxygen, are similar for the phenylalanine aromatic protons and those observed for protons in the upper portion of the acyl chain. The rates of amide-water proton exchange are fast and only slightly hindered when the peptide is bound to bicelles, indicating that the backbone does not lie within the membrane hydrocarbon. These results indicate that highly charged peptides such as the MARCKS-ED penetrate the membrane interface with aromatic amino acid side chains inserted into the hydrocarbon and the peptide backbone lying within the bilayer interface. This position may serve to enhance the electrostatic fields produced by this basic domain at the membrane interface and may play a role in the ability of the MARCKS-ED to sequester polyphosphoinositides.     

Lipocalin-type prostaglandin D2 synthase protein regulates glial cell migration and morphology through myristoylated alanine-rich C-kinase substrate: prostaglandin D2-independent effects

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.     

Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL)

The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures. We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase. We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate). The partition coefficients describing the association of unmyristoylated and myristoylated MARCKS-related protein with membranes of different phospholipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-related protein mediate the binding. However, no significant cooperativity is observed between these two domains. Interestingly, MARCKS-related protein binds to TRANSIL membranes more strongly at temperatures below their phase-transition temperature. Taking advantage of this property, MARCKS-related protein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSIL column at 4 degrees C and eluting MRP at room temperature. In conclusion, TRANSIL is a versatile tool to determine the affinity of compounds for phospholipid membranes and to purify membrane-bound proteins. TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes.     

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation.

Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.     

Regulation of the binding of myristoylated alanine-rich C kinase substrate (MARCKS) related protein to lipid bilayer membranes by calmodulin

The effector domain (ED) of MARCKS proteins can associate with calmodulin (CaM) as well as with phospholipids. It is not clear, however, whether a complex between MARCKS proteins and CaM can form at the surface of phospholipid membranes or whether CaM and membranes compete for ED binding. Using two-mode waveguide spectroscopy, we have investigated how CaM regulates the association of MARCKS-related protein (MRP) with planar supported phospholipid bilayer membranes. Bringing a solution containing CaM into contact with membranes on which MRP had previously been deposited results in low-affinity binding of CaM to MRP. A preformed, high-affinity CaM MRP complex in the aqueous phase binds much more slowly than pure MRP to membranes. Similar observations were made when a peptide corresponding to the ED of MRP was used instead of MRP. Hence CaM cannot form a stable complex with MRP once the latter is bound at the membrane surface. CaM can, however, strongly retard the association of MRP with lipid membranes. The most likely interpretation of these results is that CaM and the phospholipid membrane share the same binding region at the ED and that the ED is forced by membrane binding to adopt a conformation unfavorable for CaM binding.     

Phorbol ester-induced down-regulation of the 80-kDa myristoylated alanine-rich C-kinase substrate-related protein in Swiss 3T3 fibroblasts. Inhibition by staurosporine.

A polyclonal antiserum raised against an oligopeptide with an amino acid sequence corresponding to a sequence of the myristoylated alanine-rich C-kinase substrate (MARCKS) from mouse macrophages and rat brain recognizes the 80-kDa C-kinase substrate from Swiss 3T3 fibroblasts. Using this antiserum for quantitative determination of the 80-kDa MARCKS-related protein, we found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid down-regulation of this protein in the fibroblasts. In accordance with earlier reports, TPA causes phosphorylation of the 80-kDa protein which can be inhibited by staurosporine. Staurosporine also suppresses the TPA-induced down-regulation, possibly indicating that the down-regulation of the MARCKS-related protein is dependent on its phosphorylation by protein kinase C.     

Binding of peptides with basic and aromatic residues to bilayer membranes: phenylalanine in the myristoylated alanine-rich C kinase substrate effector domain penetrates into the hydrophobic core of the bilayer

Electrostatic interactions with positively charged regions of membrane-associated proteins such as myristoylated alanine-rich C kinase substrate (MARCKS) may have a role in regulating the level of free phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in plasma membranes. Both the MARCKS protein and a peptide corresponding to the effector domain (an unstructured region that contains 13 basic residues and 5 phenylalanines), MARCKS-(151-175), laterally sequester the polyvalent lipid PI(4,5)P2 in the plane of a bilayer membrane with high affinity. We used high resolution magic angle spinning NMR to establish the location of MARCKS-(151-175) in membrane bilayers, which is necessary to understand the sequestration mechanism. Measurements of cross-relaxation rates in two-dimensional nuclear Overhauser enhancement spectroscopy NMR experiments show that the five Phe rings of MARCKS-(151-175) penetrate into the acyl chain region of phosphatidylcholine bilayers containing phosphatidylglycerol or PI(4,5)P2. Specifically, we observed strong cross-peaks between the aromatic protons of the Phe rings and the acyl chain protons of the lipids, even for very short (50 ms) mixing times. The position of the Phe rings implies that the adjacent positively charged amino acids in the peptide are close to the level of the negatively charged lipid phosphates. The deep location of the MARCKS peptide in the polar head group region should enhance its electrostatic sequestration of PI(4,5)P2 by an "image charge" mechanism. Moreover, this location has interesting implications for membrane curvature and local surface pressure effects and may be relevant to a wide variety of other proteins with basic-aromatic clusters, such as phospholipase D, GAP43, SCAMP2, and the N-methyl-d-aspartate receptor.     

Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor.

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, has been implicated in several cellular processes, yet its physiological function remains unknown. We have studied the molecular basis of its membrane association in a cell-free system in order to help elucidate its regulation and function. First, we showed that the MARCKS protein incorporated [3H]myristate when its mRNA was translated in vitro in reticulocyte lysates. The myristoylated protein bound rapidly to freshly fractionated cell membranes, while a nonmyristoylated mutant associated to a much lesser extent (< 15% of wild type). To determine whether this binding was due to a specific cytoplasmic-face protein "receptor," as is seen with pp60v-src, we pretreated the membranes in several ways. Prior treatment of membranes with heat (100 degrees C for 3 min) or trypsin did not affect subsequent MARCKS binding. Binding was markedly decreased in 50 mM EDTA, 0.5 M NaCl, or 1.0% Triton X-100; it was restored to normal after removal of the NaCl and EDTA but was still decreased after removal of the Triton X-100. These findings argued against the existence of a protein receptor for the MARCKS protein on cellular membranes. Finally, MARCKS protein phosphorylated in vitro with protein kinase C bound to the cell membranes to the same extent as the nonphosphorylated protein; this binding was also unaffected by an excess of a synthetic peptide corresponding to the phosphorylation site domain of the protein. We conclude that, at least in this in vitro system, the membrane association of the MARCKS protein is primarily dependent on the amino-terminal myristate moiety and does not appear to involve a specific cytoplasmic-face protein receptor.     

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS)

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.     

Relationship between the major protein kinase C substrates acidic 80-kDa protein-kinase-C substrate (80K) and myristoylated alanine-rich C-kinase substrate (MARCKS). Members of a gene family or equivalent genes in different species.

Two major protein-kinase-C (PKC) substrates have been described in the literature; an 87-kDa bovine and human PKC substrate, called MARCKS, and an acidic 80-kDa PKC substrate, isolated from rat brain and Swiss 3T3 cells, termed 80K. Since there is only 66-74% sequence similarity between MARCKS and 80K, we have further investigated their relationship in this study. Southern-blot experiments with gene-specific probes demonstrated the presence of the 80K, but not MARCKS, gene in the mouse genome. Furthermore, polymerase-chain-reaction (PCR) analyses using three pairs of primers that specifically recognise either 80K, MARCKS or conserved sequences of both genes, revealed the presence of only the 80K gene in the mouse and rat genomes and only the MARCKS gene in the bovine and human genomes with mRNA expression in the corresponding brain tissues. Northern-blot analysis of a variety of tissues indicated that both 80K and MARCKS have similar patterns of expression. Most components of signal-transduction pathways are present in multiple molecular isoforms as members of a gene family. In contrast, the findings presented in this study indicate that rodent 80K and bovine and human MARCKS are not distinct members of a gene family, but represent the equivalent substrates in different species.     

Characterization of the phosphorylation sites in the chicken and bovine myristoylated alanine-rich C kinase substrate protein, a prominent cellular substrate for protein kinase C

Little is known about the important cellular substrates for protein kinase C and their potential roles in mediating protein kinase C-dependent processes. We evaluated the protein kinase C phosphorylation sites in a major cellular substrate for the kinase, a protein of apparent Mr 80,000 in bovine and 60,000 in chicken tissues; we have recently determined the primary sequences of these proteins and tentatively named them the myristoylated alanine-rich C kinase substrates. The proteins were purified to apparent homogeneity from bovine and chicken brains, phosphorylated with protein kinase C, digested with trypsin, and the phosphopeptides purified and sequenced. Four distinct phosphopeptides were identified from both the bovine and chicken proteins. Two of the phosphorylated serines were contained in the repeated motif FSFKK, one in the sequence LSGF, and one in the sequence SFK. All four sites were contained within a basic domain of 25 amino acids which was identical in the chicken and bovine proteins. All of the sites phosphorylated in the cell-free system appeared to be phosphorylated in intact cells; an additional site may have been present in the proteins from intact cells. The identity of the phosphorylation site domains from two proteins of overall 65% amino acid sequence identity suggests a potential role for this domain in the physiological function of the myristoylated alanine-rich C kinase substrate proteins.     

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.     

Thrombin-induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in bovine pulmonary artery endothelial cells

Myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate that is targeted to the plasma membrane by an amino-terminal myristoyl group. In its nonphosphorylated form, MARCKS cross-links F-actin and binds calmodulin (CaM) reciprocally. However, upon phosphorylation by PKC, MARCKS releases the actin or CaM. MARCKS may therefore act as a CaM sink in resting cells and regulate CaM availability during cell activation. We have demonstrated previously that thrombin-induced myosin light chain (MLC) phosphorylation and increased monolayer permeability in bovine pulmonary artery endothelial cells (BPAEC) require both PKC- and CaM-dependent pathways. We therefore decided to investigate the phosphorylation of MARCKS in BPAEC to ascertain whether this occurs in a temporally relevant manner to participate in the thrombin-induced events. MARCKS is phosphorylated in response to thrombin with a time course similar to that seen with MLC. As expected, MARCKS is also phosphorylated by phorbol 12-myristate 13 acetate (PMA), a PKC activator, but with a slower onset and more prolonged duration. Bradykinin also enhances MARCKS phosphorylation in BPAEC, but histamine does not. MARCKS is distributed evenly between the membrane and cytosol in BPAEC, and neither thrombin nor PMA caused significant translocation of the protein. Specific PKC inhibitors attenuated MARCKS phosphorylation by either thrombin or PMA. Since thrombin-induced MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contraction. W7, a CaM antagonist, enhances the phosphorylation of MARCKS. This was expected since CaM binding to MARCKS has been shown to decrease MARCKS phosphorylation by PKC. On the other hand, tyrosine kinase inhibitors, genistein and tyrphostin, attenuate MARCKS phosphorylation but have no effect on MLC phosphorylation, suggesting that MARCKS may be phosphorylated by kinases other than PKC. Phosphorylation of MARCKS outside the PKC phosphorylation domain would not be expected to induce the release of CaM. These data provide support for the hypothesis that MARCKS may serve as a regulator of CaM availability in BPAEC. © 1996 Wiley-Liss, Inc.     

Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP)

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein ki-nase C (PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca²⁺ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca²⁺-CaM signalling and PKC-mediated phosphorylation cascades. We have studied Ca²⁺ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca²⁺ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca²⁺ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca²⁺ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca²⁺-CaM-MRP peptide complex, as well as CD measurements of Ca²⁺-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the α-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82. Received: 26 September 1996 / 22 October 1996     

Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.     

Characterization of the targeting, binding, and phosphorylation site domains of an A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog that are encoded by a single gene.

A novel Drosophila A kinase anchor protein, Drosophila A kinase anchor protein 200 (DAKAP200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca(2+), and diacylglycerol (Li, Z., Rossi, E. A., Hoheisel, J. D., Kalderon, D., and Rubin, C. S. (1999) J. Biol. Chem. 274, 27191-27200). Experiments designed to assess this hypothesis now (a) establish the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of DAKAP200; (b) demonstrate that residues 119-148 mediate binding with Ca(2+)-calmodulin and F-actin; (c) show that a polybasic region of DAKAP200 is a substrate for protein kinase C; (d) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca(2+)-calmodulin; and (e) indicate that DAKAP200 is myristoylated and that this modification promotes targeting of DAKAP200 to plasma membrane. DeltaDAKAP200, a second product of the DAKAP200 gene, cannot tether PKAII. However, DeltaDAKAP200 is myristoylated and contains a phosphorylation site domain that binds Ca(2+)-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M(r) of 38,000 suggest that DeltaDAKAP200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. DAKAP200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca(2+), and diacylglycerol.     

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).