Major Phosphoprotein Research Articles

The organization of the intron-containing human S6 ribosomal protein (rpS6) gene and determination of its location at chromosome 9p21.

The intron-containing gene encoding human ribosomal protein S6 (rpS6), the major phosphoprotein in the mammalian ribosome, has been cloned. Using a PCR based cloning strategy we have isolated the rpS6 intron-containing gene in the presence of its many processed pseudogenes and determined the DNA sequence of the entire gene and its upstream and downstream flanking regions. The human rpS6 gene is 3979 bp in length and comprises six exons. Despite lacking a consensus TATA box, primer extension analysis indicates that the start of transcription is located at a single C residue within an 11 bp oligopyrimidine tract. The first exon, which contains the ATG start codon, is 48 bp in length. The DNA sequence in the 5' region of the gene has features of a CpG-rich island. Using fluorescence in situ hybridization (FISH) analysis the position of the rpS6 gene has been sublocalized to human chromosome 9p21. The similarities and differences between rpS6 and other previously characterized ribosomal protein genes are discussed.

The effect of -adrenergic agents and protein kinase C activators on protein phosphorylation in isolated guinea pig hearts

The incorporation of [32P]Pi into sarcolemmal, sarcoplasmic reticular and myofibrillar proteins was studied in Langendorff-perfused guinea pig hearts treated with the alpha-agonist norepinephrine or with protein kinase C activators (phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoylglycerol (D8G]. Norepinephrine was administered in the presence of propranolol and atropine, while the protein kinase C activators (PMA and D8G) were infused in the presence of propranolol, atropine and prazosin. Examination of 32P-incorporation into the various cardiac proteins revealed that there were no significant increases in the degree of phosphorylation of the: (1) 15 kDa sarcolemmal protein; (2) phospholamban in sarcoplasmic reticulum; and (3) troponin I and C protein in the myofibrils. In parallel control studies, stimulation of beating guinea pig hearts by isoproterenol was associated with a 4-5-fold increase in 32P-incorporation into phospholamban and troponin I and about a 2-fold increase in 32P-incorporation into C protein and the 15 kDa sarcolemmal protein. These findings indicate that the major cardiac regulatory phosphoproteins, which have been reported to serve as substrates for protein kinase C in vitro, are not phosphorylated by the same enzyme in perfused, beating guinea pig hearts.

The distribution and phosphorylation of the microtubule-associated protein MAP 1B in growth cones

Primary cultures of dissociated embryonic day 18 rat cerebral cortices were labelled by immunofluorescence with antibodies directed either against phosphorylated and non-phosphorylated MAP 1B (antibody 81) or against phosphorylated MAP 1B (antibody 150). Both antibodies stain cortical neurons, including their neurites and growth cones, in early (18 h) cultures, whereas only antibody 81 stained glial cells. By 4 days in culture, phosphorylated MAP 1B is largely restricted to axonal processes and growth cones, where it is often distributed in a gradient that is highest distally. In axonal processes and growth cones after 18 h and 4 days in culture, the phosphorylated form of MAP 1B is present both in a soluble form and bound to microtubules. Growth cones isolated from postnatal day 5 rat forebrain were labelled in vitro with 32P-orthophosphate and detergent soluble and insoluble (cytoskeleton) fractions prepared. SDS-PAGE analysis revealed several major phosphoproteins in isolated growth cone cytoskeletons, including MAP 1B. Phosphorylated MAP 1B was also present in the detergent soluble fraction of growth cones. Immunoblotting and immunoprecipitation with MAP 1B antibodies confirmed the identification of MAP 1B and that the protein is phosphorylated in growth cones. These data show that MAP 1B, in particular the phosphorylated isoform, is present in growth cones and suggest that phosphorylation of MAP 1B may play an important role in neurite elongation.

Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP 3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP 3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

Regulated expression of p18, a major phosphoprotein of leukemic cells.

p18 is a phosphoprotein that is present in great abundance in acute leukemia blasts and in less abundance in proliferating lymphocytes. This protein undergoes major changes in its state of phosphorylation upon induction of differentiation of leukemic cells in culture. The same protein appears to be involved in a variety of other cellular processes that include regulation of hormone secretion, T cell activation, muscle differentiation, and brain development. In this report, we describe our studies of the regulation of expression of this gene in leukemic cells. We show that the expression of this gene is markedly reduced upon induction of differentiation of a variety of leukemic cells in culture. We use a cDNA clone that we constructed earlier which encodes this protein as a probe to isolate the human chromosomal p18 gene. We characterize the 5' end of this gene in detail and identify its promoter element. We also identify a regulatory element in the first intervening sequence (IVS-1) of this gene which loses its DNase I hypersensitivity upon induction of differentiation of leukemic cells in culture. Our DNase I footprinting experiments demonstrate nuclear protein binding to multiple sequence motifs within its promoter element and its IVS-1 regulatory element. Functional studies using a transient expression system show that deletion of these sequence motifs has profound effects on the expression of this gene. These studies begin to shed some light on the mechanism of regulation of a gene that may be involved in control of cell growth and differentiation and in a variety of other vital cellular processes.

Heat shock increases turnover of 90 kDa heat shock protein phosphate groups in HeLa cells

The 90 kDa heat shock protein (hsp9O) is a major phosphoprotein which associates various other cellular polypeptides such as actin, calmodulin, steroid hormone receptors and certain protein-kinases. Little is known about the function of hsp9O in recovery from stress. In this report, we describe a dramatic increase in the rate of both phosphate uptake and dephosphorylation of hsp9O in HeLa cells submitted to acute stresses. This increased turnover of hsp9O phosphate groups might reflect a greater protein binding activity of hsp9O in stressed cells.

Effect of zinc on calmodulin-stimulated protein kinase II and protein phosphorylation in rat cerebral cortex.

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.

Insulin and Insulin-Like Growth Factors Stimulatein VivoReceptor Autophosphorylation and Tyrosine Phosphorylation of a 70K Substrate in Cultured Fetal Chick Neurons*

Insulin and insulin-like growth factors (IGFs) have been shown to regulate neuronal growth and differentiation. To investigate the possible role of tyrosine phosphorylation in the neurotrophic actions of these peptides, we examined the effects of insulin, IGF-I, and IGF-II on tyrosine phosphorylation in cultured fetal chick neurons. Tyrosine phosphorylation was detected by immunoblot analysis using antiphosphotyrosine antibodies. Under basal conditions five major phosphoproteins (170K, 140K, 115K, 103K, and 44K) and a number of minor proteins were detected by three separate antisera. In response to insulin, IGF-I, or IGF-II, an 87K membrane-associated protein (pp87) became phosphorylated on tyrosine in a rapid, dose-dependent manner. The Mr of pp87 was identical to that of the insulin and IGF-I receptor beta-subunits. Comparison of the dose-response curves for pp87 phosphorylation by insulin and IGF-I suggests that each peptide stimulated autophosphorylation of its own receptor beta-subunit. The maximal response obtained with IGF-I was approximately 10-fold higher than that obtained with insulin (3- to 5-fold), consistent with the larger number of IGF-I receptors in these cells. The maximal response obtained with IGF-II was 4- to 7-fold higher than that with insulin, suggesting that it acts through the insulin receptor as well. Tyrosine that IGF-II acts through the insulin receptor as well. Tyrosine phosphorylation of other proteins by the activated receptor kinases was not detected in neurons cultured for 5 days, however neurons cultured for only several h contained a predominant 70K protein (pp70) that was phosphorylated on tyrosine in response to all three hormones. Tyrosine phosphorylation of pp70 was maximal after 2-5 h in culture and was undetectable in neurons cultured longer than 24 h. The time course and dose dependence of pp70 phosphorylation in response to hormone paralleled that of insulin and IGF-I receptor autophosphorylation. These results demonstrate that both insulin and IGF-I receptors are active tyrosine kinases in fetal neurons, and that pp70 represents a potential endogenous substrate for the insulin and IGF-I receptor kinases. The transient phosphorylation of pp70 may be involved in the neurotrophic effects of insulin and insulin-like growth factors.

Characterization of the pp70 protein phosphorylation in the extract of rice young panicle

The endogenous phosphorylation pattern of the extract prepared from rice young panicle has been examined. The extract was phosphorylated in vitro by incubating with [γ-32p] ATP, and then analyzed by SDS-PAGE and autoradiography. At least eight major phosphoproteins with apparent molecular weights of 70, 60, 52, 40, 33, 30, 20, 16 and 15kd were detected in the crude extract of rice young panicle. The pp70 which represents the major phosphoprotein in the crude extract of young panicle of rice is a cytosolic protein. Photoaffinity labeling with 8-azido-ATP revealed that a major protein with molecular weight 42,000 dalton but not pp70 can be specifically bound ATP. This suggests that pp70 is not a protein kinase itself, but a substrate of protein kinase. The in vitro phosphorylation of the pp70 occurs at a serine or threonine residue and is time and temperature dependent. The phosphorylation of pp70 does not depend by exogenous Ca ++ or cAMP, suggesting that pp70 is a major substrate of an Ca ++ or cAMP independent protein kinase in rice young panicle.

Characterization of a cDNA for chicken osteopontin: expression during bone development, osteoblast differentiation, and tissue distribution

The chicken bone phosphoprotein (approximately 66-kDa BPP) is a major noncollagenous component of bone and is the major phosphoprotein synthesized by cultured chicken embryo osteoblasts [Gotoh, Y., Gerstenfeld, L. C., & Glimcher, M. J. (1990) Eur. J. Biochem. 87, 49-58]. A cDNA clone for this protein was isolated from an expression library made from embryonic chicken bone mRNA. The complete primary protein sequence of 264 amino acids was deduced from the cDNA sequence inclusive of a 16 amino acid signal peptide sequence and terminated by 4 in-frame stop sequences. A sequence alignment indicated an approximate 35% overall similarity in protein sequence between the avian approximately 66-kDa BPP and the mammalian protein osteopontin, while at the nucleotide level 60% similarity was observed. Features of this sequence which showed the greatest similarity to mammalian osteopontin included a region in which seven of nine consecutive residues are aspartic acid, a recognition sequence for integrin-mediated cell binding (-Arg-Gly-Asp), and four possible recognition sequences for phosphorylation by casein kinase II. Hybridization analysis indicated a message of 1.5 kb found predominantly in bone and kidney. The mRNA was inducible in phorbol ester treated primary cultures of chondrocytes which show no expression under normal growth conditions. A temporal induction was seen during osteoblastic differentiation both in vivo and in vitro, thus suggesting that regulation of the approximately 66-kDa BPP is under transcriptional control during osteoblast development. In summary, both the protein's primary structure and its biological features suggest that it is the avian homologue to mammalian protein osteopontin.

Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor.

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.

Characterization of protein-tyrosine kinase activity in the canine prostate

Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44-63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase.

Ontogenetic studies on electrophoretic patterns of prealbumin proteins in the chicken. Comparison of egg yolk and blood plasma of embryos, chicks and adults.

The electrophoretic patterns of chicken prealbumin proteins were compared among egg yolk and blood plasma of embryos, chicks and adults with emphasis on the estrogen-dependent proteins. Chicken yolk phosvitin, a major phosphoprotein, was detected in the slowest migrating prealbumin-1 zone stained with rhodamine B.Pa-2, phosphorus-free estrogen-dependent protein, was found in egg yolk and plasma of laying hens, 20-day-embryo, and 1-day-old chicks, but not detected in the plasma of 16-week-old immature cockerels and pullets and mature cocks.

Protein phosphorylation in guinea-pig myenteric ganglia and brain: Presence of calmodulin kinase II, protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The α subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the β subunit by autophosphorylation. The ratio of β to α subunit was considerably higher in ganglia than in brain and ganglia β subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinases and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.

Partial characterization and subunit analysis of major phosphoproteins of egg yolk in the fish, Oryzias latipes

1. 1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka ( Oryzias latipes) eggs were performed. 2. 2. PP1 ( M r ≈ 210,000) and PP2 ( M r ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively. 3. 3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ. 32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively. 4. 4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.

Modulation of endogenous protein phosphorylation in plant mitochondria by respiratory substrates

Purified mitochondria from potato (Solanum tuberosum L. cv. Bintje) tubers were incubated with [γ32P]‐ATP, respiratory substrates and various effectors. The total incorporation of 32P into proteins was measured and the phosphoprotein pattern investigated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and autoradiography. Total incorporation was strongly reduced (by 70–80%) by the respiratory substrates, succinate, pyruvate and NADH. The half‐maximal inhibition was at 0.03, 0.3, and 0.3 mM, respectively. The labelling of the major phosphoproteins of 40 and 42 kDa (probably both the α‐subunit of the pyruvate dehydrogenase, EC 1.2.4.1) as well as of minor polypeptides of 26–33 kDa was reduced. A concomittant increase in the labelling of the 14 and 16 kDa bands occurred in the presence of succinate in fall but this increase could not be detected in late winter. The reduction in total labelling caused by NADH and succinate was unaffected by changes in the membrane potential (e.g. addition of uncouplers) or by inhibition of electron transport (e.g. by KCN). Malonate inhibition of succinate oxidation reversed the effects of succinate on labelling‐ The mechanism(s) by which respiratory substrates might affect protein kinase activity is discussed.

Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes.

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.

Vinculin is one of the major endogenous substrates for intrinsic tyrosine kinases in neuronal growth cones isolated from fetal rat brain

Neuronal growth cones, the motile tips of growing neuronal processes, are responsible for the exact guidance of extending neurites. To elucidate the mechanisms of their biochemical signal transduction in growth cones, the growth-cone-enriched fraction was isolated biochemically from fetal rat brain and the endogenous protein phosphorylation in the fraction was analyzed under the conditions where tyrosine residues were preferentially phosphorylated. One of the major phosphoproteins was a 130-kDa slightly acidic protein which reacted with antiphosphotyrosine antibody. Its phosphoryl residues were alkali-stable. Thus, the 130-kDa protein was concluded to be susceptible to tyrosine phosphorylation. This protein was a component of cytoskeletal proteins thought to be associated indirectly with membranes. All the behavior of the 130-kDa protein was compatible with the properties of vinculin, a component of focal contacts which are responsible for the stable or motile adhesion between cells or between a cell and the substratum. Immunochemical analyses showed that the 130-kDa protein was specifically recognized by anti-vinculin antibody. Therefore, the 130-kDa protein was concluded to be vinculin. Tyrosine phosphorylation of the protein appeared to be relatively more pronounced in the growth-cone-enriched fraction than in adult synaptosomes. The results suggest that tyrosine phosphorylation of vinculin may be regulated developmentally and it may be involved in the functions of growth cones.