Detergents and Peptides Alter Proteolysis and Calmodulin Binding of B-50/GAP-43 In Vitro

Abstract: The neuronal growth-associated protein B-50/GAP-43 is a substrate for protein kinase C, binds to calmodulin in a calcium-independent manner, and in vitro is subject to an endogenous and chymotrypsin-mediated hydrolysis in the vicinity of the single kinase C phosphorylation site. All of these processes can be influenced by corticotrophin (ACTH). In the present study we have investigated whether these biochemical interactions involving B-50 could have common structural determinants. Chymotryptic digestion of B-50 in the presence or absence of a nonionic detergent and ACTH demonstrated that hydrolysis is potentiated by a lipid-like environment that primarily affects the protein rather than the protease or the peptide. Furthermore, this lipid dependency appears to extend to the binding of dephosphorylated B-50 to calmodulin, which appears to occur only in the presence of a nonionic detergent or lipid and the absence of calcium. A structure-activity study for ACTH-mediated inhibition of B-50 proteolysis by an endogenous protease that copurifies with B-50 in a detergent extract of synaptosomal plasma membranes showed that ACTH_1–24, ACTH_5–24, ACTH_5–16, dynorphin, and corticostatin inhibited the conversion of rat B-50 to B-50_41–226. In contrast, ACTH_7–16, Org₂₇₆₆, and neurotensin had no detectable effect on B-50 proteolysis at concentrations of 10 and 50 µM. The results indicate that in common with effects in other B-50-containing systems, inhibition of proteolysis is related to the presence of a basic amphiphilic helix in those ACTH fragments and analogues that were inhibitory and, moreover, the presence of this motif in other peptides appears to confer inhibitory activity. The results are discussed with reference to the putative secondary structure of B-50 and changes that may take place in the presence of membrane lipids or nonionic detergents. The conclusions of this study suggest that in vitro B-50 is subject to regulation by posttranslational enzymes and binding proteins as a consequence of its ability to adapt an amphiphilic helix conformation.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg²⁺ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.     

A Radioimmunoassay for the Phosphoprotein B-50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.     

Determination of Changes in the Phosphorylation State of the Neuron-Specific Protein Kinase C Substrate B-50 (GAP43) by Quantitative Immunoprecipitation

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.     

Purification and Some Characteristics of an ACTH-Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane-bound protein (B-50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X-100 in 75 mM-KCl. Using column chromatography over DEAE-cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B-50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B-50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B-50 after 60 min. The inhibition by ACTH_{1_24} of the B-50 protein kinase in ASP 55–80 was dose-dependent; the half-maximal effective concentration was 5 × 10⁻⁶ M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor-phins had no effect on the B-50 protein kinase. The B-50 protein kinase required both magnesium and calcium for optimal activity. Using two-dimensional electrophoresis on polyacrylamide slab gels the B-50 protein kinase and the B-50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B-50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B-50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B-50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.     

Affinity-Purified Anti-B-50 Protein Antibody: Interference with the Function of the Phosphoprotein B-50 in Synaptic Plasma Membranes

Abstract: Affinity-purified anti-B-50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B-50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B-50 protein and its adrenocorticotropin-sensitive protein kinase, obtained from rat brain. Anti-B-50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B-50 protein isolated by an improved procedure. The purified antibodies reacted only with the B-50 and B-60 protein, a proteolysis derivative (of B-50), as assessed by the sodium dodecyl sulfate-gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B-50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4-phosphate was not altered. Inhibition by ACTH _1–24 of the endogenous phosphorylation of B-50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-diphosphate. These data support our hypothesis on the functional interaction of B-50 protein and phosphatidylinositol 4-phosphate kinase in rat brain membranes. The evidence shows that purified anti-B-50 protein antibodies can be used to probe specifically the function of B-50 protein in membranes.     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

ACTH,cyclic nucleotides,and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated³²P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH_1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10^–10 to 10^–4 M. In a number of protein bands, a biphasic effect of ACTH_1–24 was observed: in concentrations of 10^–4–10^–5 M, a reduced amount of³²P was found; in concentrations of 10^–6–10^–7 M, hardly any effect could be detected, whereas consistently at concentrations around 10^–8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

ACTH1–24 Releases a Protein from Synaptosomal Plasma Membranes

Abstract: Brain membranes contain several protein kinases, all of which appear to play a role in the regulation of neuronal functioning. These membranes also contain numerous (phospho) proteins. It has been proposed that the degree of phosphorylation of some of these proteins may affect neuronal membrane properties. In a series of previous reports we showed that ACTH_1-24 inhibits the endogenous phosphorylation of several synaptosomal plasmamembrane (SPM) proteins including the B-50 protein. Although we have speculated that the degree of phosphorylation of B-50 may be important in regulating the turnover of membrane (poly)-phosphoinositides, the exact nature of the interaction between ACTH_1-24 and B-50/B-50 protein kinase is unknown. The purpose of the present study was to determine whether treatment of SPM with ACTH_1-24 will lead to a specific release of proteins from SPM. We found that ACTH_1-24 specifically releases a 41,000 M_r protein from rat brain SPM. Although we are not certain about the biological significance of the release of this polypeptide, it is of sufficient interest for further research in view of the lack of success of finding binding of labeled ACTH to brain membranes.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.     

Localization in the synaptic junction of the cyclic AMP stimulated intrinsic protein kinase activity of synaptosomal plasma membranes.

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggests that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Localization in the synaptic junction of the cyclic amp stimulated intrinsic protein kinase activity of synaptosomal plasma membranes

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggest that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.     

4-Aminopyridine inhibits synaptosomal plasma membrane protein phosphorylation in vitro: effect of the selective NMDA-antagonist 2-amino-5-phosphonovalerate

Phosphorylation of synaptosomal plasma membranes from rat hippocampus in the presence of the convulsant drug 4-aminopyridine resulted in the inhibition of the phosphorylation of the nervous tissue specific protein kinase C substrate protein B-50 (48 kDa) and the alpha-subunit of calcium/calmodulin-dependent protein kinase II (50 kDa). Preincubation of SPM with 2-amino-5-phosphonovalerate prevents the inhibition of B-50 phosphorylation by 4-aminopyridine, but had no effect on the inhibition of 50 kDa phosphorylation. 2-Amino-5-phosphonovalerate is known to be a specific N-methyl-D-aspartate antagonist and has anti-epileptic activity in vitro and in vivo. Several other anti-epileptic drugs tested did not influence the 4-aminopyridine-induced inhibition of protein phosphorylation.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.