Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells.

Activation of the extracellular signal-regulated protein kinase (ERK) cascade by membrane-type-1 matrix metalloproteinase (MT1-MMP).

The mechanisms underlying membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent induction of cell migration were investigated. Overexpression of MT1-MMP induced a marked increase in cell migration, this increase being dependent on the presence of the cytoplasmic domain of the protein. MT1-MMP-dependent migration was inhibited by a mitogen-activated protein kinase kinase 1 inhibitor, suggesting the involvement of the extracellular signal-regulated protein kinase (ERK) cascade in the induction of migration. Accordingly, MT1-MMP overexpression induced the activation of ERK, this process being also dependent on the presence of its cytoplasmic domain. MT1-MMP-induced activation of both migration and ERK required the catalytic activity of the enzyme as well as attachment of the cells to matrix proteins. The MT1-MMP-dependent activation of ERK was correlated with the activation of transcription through the serum response element, whereas other promoters were unaffected. Taken together, these results indicate that MT1-MMP trigger important changes in cellular signal transduction events, leading to cell migration and to gene transcription, and that these signals possibly originate from the cytoplasmic domain of the protein.

Matrix proteinase inhibition by AE-941, a multifunctional antiangiogenic compound.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in tissue remodeling under normal physiological and pathological conditions and are thus attractive targets for both diagnostic and therapeutic purposes. Here, we examined the effect of AE-941, an orally bioavailable standardized extract made of cartilage that shows significant antiangiogenic and antimetastatic properties in vivo, on the activity of various members of the MMP family. MATERIALS AND METHODS: The effect of AE-941 on the activity of MMPs was assessed by fluorimetric assays and by substrate gel zymography. RESULTS: AE-941 markedly inhibits the gelatinolytic activity of MMP-2 and to a lesser extent those of MMP-1, MMP-7, MMP-9 and MMP-13. AE-941 also inhibited the elastinolytic activities of MMP-2 and MMP-9 as well as MMP-12 (metalloelastase), porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). Western blot analysis revealed the presence within AE-941 of immunoreactive TIMP-like proteins, suggesting that these proteins may be at least partly responsible for the observed MMP inhibition. CONCLUSIONS: Taken together, these results demonstrate that AE-941 contains TIMP-like proteins that could be responsible for the specific inhibition of MMPs. Given the recent studies suggesting the presence within this compound of specific inhibitor(s) of endothelial cell proliferation, AE-941 appears as a pleotropic agent able to interfere with several biochemical steps leading to angiogenesis and to other physiopathological conditions. Since AE-941 is currently under Phase III clinical investigations, these findings are also of considerable importance for our understanding of its anticancer properties.

Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43 kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1 had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.

AE-941 (Neovastat): a novel multifunctional antiangiogenic compound.

AE-941 (Neovastat) is a naturally occurring product extracted from cartilage and has antiangiogenic properties. It has reached Phase III clinical trial evaluation for the treatment of solid tumors (non-small cell lung cancer and renal cell carcinoma) and a pivotal Phase II clinical trial in multiple myeloma is ongoing. AE-941 inhibits several steps of the angiogenesis process, including matrix metalloproteinase activities and VEGF signaling pathways. Moreover, AE-941 induces endothelial cell apoptosis and tissue-type plasminogen activator activity, thus suggesting that it is a multifunctional antiangiogenic drug. Results from Phase I/II clinical trials indicate that AE-941, given orally, is well tolerated. Moreover, the median survival time in patients with renal cell carcinoma and non-small cell lung cancer was significantly longer in patients receiving high doses of AE-941 compared to low doses.

Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism.

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Tyrosine phosphorylation of the vascular endothelial-growth-factor receptor-2 (VEGFR-2) is modulated by Rho proteins.

The effects of Rho-specific modifying toxins on the tyrosine phosphorylation of endothelial cell proteins were investigated. Incubation of the cells with the Rho-activating toxin cytotoxic necrotizing factor 1 (CNF1) induced a marked increase in the tyrosine phosphorylation of a number of signalling intermediates of the vascular endothelial growth factor (VEGF)-mediated cascade, including focal adhesion kinase, paxillin, phospholipase Cgamma1 and a Shc-associated protein of 195 kDa. Both CNF1- and VEGF-dependent tyrosine phosphorylation of these proteins were significantly reduced by prior incubation with C3 transferase, a known inhibitor of RhoA function, suggesting a Rho-dependent mechanism. The stimulation of endothelial cells with CNF1 resulted in a marked increase in the tyrosine phosphorylation of the VEGF receptor (VEGFR)-2, which was correlated with a stimulation of its kinase activity and with its association with downstream tyrosine phosphorylated proteins. The stimulatory effect of CNF1 was specific for VEGFR-2 since the phosphotyrosine content of VEGFR-1 was not affected by the toxin. Transient overexpression of a dominant-active RhoA mutant also induced an increase in the tyrosine phosphorylation of the VEGFR-2, whereas overexpression of a dominant-inactive form of the protein was without effect. Taken together, these results indicate that Rho proteins may play an important role in angiogenesis by modulating the tyrosine phosphorylation levels of VEGFR-2.

Matrix metalloproteinase inhibition by green tea catechins.

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.

P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries.

A significant proportion of P-glycoprotein (P-gp) and caveolin was co-localized in caveolae isolated from resistant (CH(R)C5) cells overexpressing P-gp and from drug-sensitive Chinese hamster ovary cells (AuxB1). The proportion of P-gp and caveolin associated with caveolar microdomains was higher in CH(R)C5 cells grown in the presence of P-gp substrates (cyclosporin A or colchicine) than in untreated CH(R)C5 cells. Coimmunoprecipitation of P-gp and caveolin from CH(R)C5 lysates suggests that there is a physical interaction between them. Furthermore, co-localization of P-gp and caveolin was found in caveolae from brain capillaries, indicating that this association also takes place in vivo.

Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills?

The use of crude cartilage for the treatment of human cancers remains a subject of controversy. In this brief commentary, we reviewed the current knowledge on the anticancer properties of cartilage. We then presented the properties of AE-941, a novel standardized water-soluble extract derived from shark cartilage that represents less than 5% of the crude cartilage. It is a multifunctional antiangiogenic product that contains several biologically active molecules. EA-941 is one of the few antiangiogenic drugs that is under phase III clinical investigation. It is currently evaluated in Europe and North America for the treatment of refractory renal cell carcinoma and in North America for metastatic non small cell lung cancer.

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Purification, cloning, and sequence analysis of a Mr = 30,000 protein from sea urchin axonemes that is important for sperm motility. Relationship of the protein to a dynein light chain.

We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immunoblots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility.

Cytosolic proteins of 21-23 kDa are methylated by kidney cortex membrane-associated C-terminal carboxyl methyltransferases.

We have studied the effect of a soluble fraction from kidney cortex on the C-terminal carboxyl methylation of 21-23 kDa proteins catalyzed by membrane-associated methyltransferases. Addition of soluble proteins to isolated luminal, antiluminal and intracellular membranes resulted in a large increase in the methylation of the membrane-associated 21-23 kDa substrates. Fractionation of the soluble extract from the cortex by Q-Sepharose anion exchange chromatography showed the presence of two distinct peaks of proteins presenting stimulating activities, eluting at 0.15 M (peak I) and 0.4 M (peak II) NaCl, respectively. Both peaks eluted as proteins of apparent molecular sizes of 40 kDa upon Superose 6 gel-filtration chromatography. No methylation activity towards N-acetyl-S-trans,trans-farnesylcysteine (AFC), a good substrate for C-terminal carboxyl methyltransferases, was associated with either peaks. In contrast, the increase in methylation induced by these proteins was strongly inhibited by AFC, suggesting that the methylation induced by these factors occurred on C-terminal isoprenylated cysteine residues. Both partially purified proteins competitively inhibited the methylation of AFC by the membrane-associated enzymes, suggesting that they may represent substrates for the methyltransferases. Immunoblotting of these partially purified soluble substrates with a rabbit polyclonal antibody directed against the small G-protein CDC42 showed the presence of this protein in peak I but not in peak II. Taken together, these results suggest the presence in a soluble fraction from the kidney of distinct methyl-accepting proteins, one of these being tentatively identified as the small G-protein CDC42.

Asymmetrical distribution of L-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex.

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

Subcellular distribution and guanine nucleotide dependency of COOH-terminal methylation in kidney cortex.

The subcellular distribution of COOH-terminal carboxyl methyltransferase and methylated substrates was studied in purified brush-border and basolateral plasma membranes, as well as in crude intracellular membranes and the cytosolic fraction isolated from rat kidney cortex. The three membrane fractions showed intrinsic carboxyl methylation of 21- to 23-kDa proteins, whereas 18- and 41-kDa methylated proteins were observed in the cytosol. In contrast, methylation activities toward N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic farnesylated substrate, were found to be strictly associated with membranes, with no detectable level of activity in the cytosol. Methylation of all membrane-associated substrates was inhibited by AFC but remained unaffected by TS-isoD-YSKY, a synthetic isopeptide recognized by L-isoaspartyl methyltransferase, suggesting that the membrane-associated substrates were methylated on a COOH-terminal isoprenylated cysteine residue. The membrane-associated methylated proteins were tightly bound to the membranes as reflected by their extraction with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate but not with 1 M NaCl or 2 M urea. The nonhydrolyzable analogues of GTP and GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), markedly increased the methylation of the 21- to 23-kDa substrates, whereas ATP gamma S and ADP beta S were without effect. This effect of guanine nucleotides was restricted to endogenous 21- to 23-kDa substrates with no stimulation of methylation of the exogenous substrate, AFC. Our results show a wide distribution of both COOH-terminal protein carboxyl methyltransferase activities and associated methylated substrates in the kidney cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a membrane-bound protein carboxyl methyltransferase from rat kidney cortex.

Class II protein carboxyl methyltransferases (EC 2.1.1.77) are known to exist predominantly in a soluble form in all cells studied so far. These enzymes have been purified to homogeneity from the cytosols of many mammalian tissues but not from membranes. We describe here the purification to apparent homogeneity of a membrane-associated protein carboxyl methyltransferase from the brush border membrane of rat kidney. The enzyme was purified by fast protein liquid chromatography on Superdex 75 and Mono-Q and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of a single 27,300 polypeptide. The purified enzyme recognizes exogenous substrate proteins such as ovalbumin and gamma-globulins as well as synthetic peptides containing a L-isoaspartyl residue but not a synthetic peptide containing a farnesylated C-terminal cysteine (S-farnesyl-LARYKC). The Km for S-adenosyl-L-methionine with ovalbumin as the substrate is 1.5 microM and the purified enzyme is sensitive to inhibition by S-adenosyl-L-homocysteine (Ki = 0.3 microM). Peptide map obtained after Staphylococcus aureus V8 protease digestion of brush border membrane protein carboxyl methyltransferase showed a fragmentation pattern that was identical to that obtained for a soluble protein carboxyl methyltransferase purified according to the same procedure, indicating a high degree of homology. These results support the notion that class II protein carboxyl methyltransferases are not restricted to a cytosolic localization and show that the membrane-bound form of this enzyme shares many characteristics with known cytosolic protein carboxyl methyltransferases.

Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.

We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.