Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP

We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis.     

Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues

Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues.     

Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells.

Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Molecular regulation of phagocyte function. Evidence for involvement of a guanosine triphosphate-binding protein in opsonin-mediated phagocytosis by monocytes

Although many functions of phagocytes are known to be regulated by guanosine triphosphate (GTP)-binding proteins, phagocytosis itself has not been considered one of these. However, previous studies have examined only unstimulated neutrophil phagocytosis. Motivated by our previous work, which showed that stimulated neutrophil phagocytosis is regulated by GTP-binding proteins (H. D. Gresham, M. G. Peters, and E. J. Brown. 1986. J. Cell Biol. 103:215a), we have examined the effect of pertussis toxin (PT) on monocyte receptor-mediated phagocytosis. PT inhibited unstimulated and fibronectin-stimulated IgG-mediated phagocytosis and also inhibited C3b-mediated phagocytosis stimulated by fibronectin or phorbol dibutyrate. Cholera toxin (CT) had no effect on unstimulated or stimulated phagocytosis mediated by IgG or C3b. PT inhibition of phagocytosis was not mediated via increases in cellular cAMP levels or by inhibition of the respiratory burst. Inhibition of phagocytosis did not result from decreased numbers of plasma membrane opsonin receptors nor decreased ability to bind opsonized targets. Although phorbol ester-stimulated phagocytosis was inhibited by PT, ligand-independent internalization of CR1 stimulated by phorbol dibutyrate proceeded normally in PT-intoxicated cells. We conclude that a PT-sensitive GTP-binding protein does regulate phagocytic function in monocytes. This protein operates on a molecular mechanism specific to the process of ingestion in both unstimulated monocytes and in cells stimulated to increase phagocytosis. Because unstimulated neutrophil phagocytosis is unaffected by PT or CT, and stimulated neutrophil phagocytosis is inhibited by both PT and CT, our data also demonstrate that monocytes and neutrophils have distinct mechanisms for regulation of phagocytic function.     

Involvement of phage T5 tail proteins and contact sites between the outer and inner membrane of Escherichia coli in phage T5 DNA injection.

The penetration of phage T5 DNA into the Escherichia coli envelope takes place through ion channels (Boulanger, P., and Letellier, L. (1992) J. Biol. Chem. 267, 3168-3172). To identify putative phage protein(s) involved in the formation of these channels, E. coli cells were infected at 37 degrees C with radioactively labeled phage and their envelopes were fractionated. After a flotation gradient, proteins belonging to the phage tail were recovered both in fractions containing the contact sites between the inner and outer membranes and in the outer membrane. The electrophoretic banding pattern of phage proteins indicates that the contact sites were enriched in the protein pb2. Moreover, infected cells were significantly enriched in contact sites as compared to intact cells. There was no enrichment of contact sites and very little radioactivity was found in this fraction and in the outer membrane when the cells were infected at 4 degrees C (i.e. under conditions where the phage does not inject its DNA). These results suggest that both contact sites and pb2 may play a central role in the translocation of phage T5 DNA.     

Retinoids stimulate the release of superoxide by neutrophils and change their morphology

All-trans-retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 +/- 14 SD and 53 +/- 5 SD nmol of O2-/min/10(7) cells, respectively. Superoxide release by unstimulated cells was negligible. All-trans-retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis-unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870-7877) are discussed in light of possible mechanisms.     

Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro

Two intermediary kinases in a protein serine/threonine kinase cascade that is triggered in the response of Swiss 3T3 cells to epidermal growth factor (EGF) have been identified. Several separable EGF-stimulated serine/threonine kinase activities were characterized in the preceding paper (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E.G. (1990) J. Biol. Chem. 265, 11487-11494). These were preincubated in various combinations in the presence of MgATP with chromatographic fractions from unstimulated cell extracts. Activation of the rate of phosphorylation of a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was observed on preincubation of the breakthrough fraction from unstimulated cell extracts with either of two distinct EGF-stimulated kinase activities, each of which phosphorylated myelin basic protein. Kinetic analysis and fractionation by sizing gel chromatography demonstrated that two myelin basic protein kinase activities (of approximately 30 and approximately 50 kDa) represented the activating components in the mixtures whereas the unstimulated cell extract breakthrough gave rise in each case to the activated Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala peptide kinase activity of approximately 110 kDa. Inasmuch as the in vitro activation reactions required magnesium plus ATP and were reversed by protein phosphatase treatment, an activation mechanism involving phosphoryl transfer is suggested.     

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.     

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.     

Isolation of stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian heart devoid of inhibitory modulator of adenosine 3':5'-monophosphate-dependent protein kinase.

The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.     

Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.     

Differential stability of biogenesis intermediates reveals a common pathway for aquaporin-1 topological maturation

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.     

Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations.

Mitogen-activated protein kinase (p42mapk) becomes transiently activated after treatment of serum-starved murine Swiss 3T3 cells or EL4 thymocytes with a diversity of mitogens. Similarly, a meiosis-activated protein kinase (p44mpk) becomes stimulated during maturation of sea star oocytes induced by 1-methyladenine. Both p42mapk and p44mpk have been identified as protein-serine/threonine kinases that are activated as a consequence of their phosphorylation. Because homologous protein kinases may play essential roles in both mitogenesis and oogenesis, we have compared in detail the biochemical properties of these two kinases. We find that these kinases are highly related based on their in vitro substrate specificities, sensitivity to inhibitors, and immunological cross-reactivity. However, they differ in apparent molecular weight and can be separated chromatographically, indicating that the two enzymes are distinct. Furthermore, in the course of this investigation, we have identified a 44-kDa protein kinase in mitogen-stimulated Swiss mouse 3T3 cells and EL4 thymocytes that co-purifies with p44mpk and thus appears to be a closer homolog of the sea star enzyme. Analysis of these protein kinases clarifies the relationships between a set of tyrosine-phosphorylated 41-45-kDa proteins present in mitogen-stimulated cells (Martinez, R., Nakamura., K. D., and Weber, M. J. (1982) Mol. Cell. Biol. 2, 653-655; Cooper, J. A., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37), two myelin basic protein kinases identified in epidermal growth factor-treated Swiss mouse 3T3 cells (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494), and p42mapk. Our work points to the existence of a group of related serine/threonine protein kinases, regulated by tyrosine phosphorylation and functioning at different stages of the cell cycle.     

The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells

Human nasopharyngeal epidermoid carcinoma (KB) cells contain a membrane-associated particulate folate-binding protein which is important in the cellular accumulation of physiologic folates (Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, P. C., and Kolhouse, J. F. (1985) J. Biol. Chem. 260, 14911-14917) and in the binding of methotrexate (Kane, M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) J. Biol. Chem. 261, 44-49). A soluble folate-binding protein appears in media exposed to proliferating KB cells. We have purified to homogeneity both the membrane-associated and the soluble folate-binding proteins from the KB cell tissue culture system. The purified membrane-associated and soluble folate-binding proteins give single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr values of 50,000 and 40,000, respectively. The membrane-associated folate-binding protein contains 45,000 g of amino acids and the soluble folate-binding protein contains 24,000 g of amino acids per mole of folate bound. Each of the purified proteins has a single folate-binding site, and the carbohydrate content is approximately 25% for each species of protein. The affinity constants for 5-methyltetrahydrofolate of the membrane-associated and soluble folate-binding proteins are 0.3 and 2.5 X 10(9) liters/mol, respectively. The affinities of various polyglutamated forms of methotrexate are similar for each protein, increase as the chain length of the polyglutamate increases (from approximately 0.004 X 10(9) liters/mol for methotrexate to 0.3 X 10(9) liters/mol for methotrexate heptaglutamate), are equal to the affinity for 5-methyltetrahydrofolate, and exceed the reported increase in affinity of methotrexate polyglutamates for dihydrofolate reductase.     

A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.     

Calcium influx in a rat mast cell (RBL-2H3) line. Use of multivalent metal ions to define its characteristics and role in exocytosis

An increase in concentration of cytosolic Ca2+ ([Ca2+]i) is associated with an accelerated influx of 45Ca2+ when cultured RBL-2H3 cells are stimulated with either antigen or analogs of adenosine although these agents act via different receptors and coupling proteins (Ali, H., Cunha-Melo, J.R., Saul, W.F., and Beaven, M.A. (1990) J. Biol. Chem. 265, 745-753). The same mechanism probably operates for basal Ca2+ influx in unstimulated cells and for the accelerated influx in stimulated cells. This influx had the following characteristics. 1) It was decreased when cells were depolarized with high external K+; 2) it was blocked by other cations (La3+ greater than Zn2+ greater than Cd2+ greater than Mn2 = Co2+ greater than Ba2+ greater than Ni2+ greater than Sr2+) either by competing with Ca2+ at external sites (e.g. La3+ or Zn2+) or by co-passage into the cell (e.g. Mn2+ or Sr2+); and 3) the inhibition of influx by K+ and the metal ions had exactly the same characteristics whether cells were stimulated or unstimulated even though influx rates were different. The dependence of various cellular responses on influx of Ca2+ was demonstrated as follows. The stimulated influx of Ca2+, rise in [Ca2+]i, and secretion, could be blocked in a concentration-dependent manner by increasing the concentration of La3+, but concentrations of La3+ (greater than 20 microM) that suppressed influx to below basal rates of influx markedly suppressed the hydrolysis of inositol phospholipids (levels of inositol 1,4,5-trisphosphate were unaffected). Some metal ions, e.g. Mn2+ and Sr2+, however, supported the stimulated hydrolysis of inositol phospholipid and some secretion in the absence of Ca2+. Thus a basal rate of influx of Ca2+ was required for the full activation of inositol phospholipid hydrolysis, but in addition an accelerated influx was necessary for exocytosis.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).     

The transport of proteins into yeast mitochondria. Kinetics and pools

By double isotope pulse-labeling of yeast cells, we determined the kinetics of labeling at 9 degrees C of total mitochondrial membrane, mitochondrial matrix, and cytosolic proteins, the alpha, beta, and gamma subunits of F1 ATPase, and glyceraldehyde-3-phosphate dehydrogenase. We find that none of the mitochondrial proteins show a lag in the incorporation of label compared to cytosolic proteins. These results argue against the existence in the cytosol of large pools of mitochondrial proteins awaiting transport into the organelle. Cycloheximide addition during the pulse stops [35S]methionine incorporation into mitochondrial membrane and cytosolic proteins rapidly (approximately 1 min) and with identical kinetics. Compared to cytosolic protein, however, there is a persistent incorporation of label into mitochondria after a chase with cold methionine (t1/2 approximately 1.5 min at 9 degrees C) which cannot be accounted for solely by chain completion. We conclude that this continued incorporation reflects some transport process in addition to a completion of a round of translation. When cells are labeled during a synchronous "restart" of protein synthesis, where ribosome run-off from mRNA was first induced either by incubating cells for 4 h at 0 degrees C or by treatment with 5 mM aurintricarboxylic acid, the initial rate of incorporation of label into mitochondrial protein now lags behind that of cytosolic proteins. From these results and those in the accompanying report (Ades, I.Z., and Butow, R.A. (1980) J. Biol. Chem. 255, 9918-9924) we propose that the translation of mRNA specific for mitochondrial proteins takes place in the cytoplasm and that at least a portion of the polysomes are then transported and bind to the outer mitochondrial membrane, followed by completion of translation and transfer of the newly synthesized polypeptides into the mitochondria. From a consideration of all of the available data on protein transport into mitochondria in yeast, we conclude that cytoplasmic polysomes bound to the outer mitochondrial membrane function in the transport of proteins into mitochondria by a process not necessarily mutually exclusive of post-translational transport.