Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.

Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.     

Differential Phosphorylation of Myelin-Associated Glycoprotein Isoforms in Cell Culture

The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP. The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG. 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.     

The amino acid sequences of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily

The myelin associated glycoproteins (MAG) are integral plasma membrane proteins which are found in oligodendrocytes and Schwann cells and are believed to mediate the axonal-glial interactions of myelination. In this paper we demonstrate the existence in central nervous system myelin of two MAG polypeptides with Mrs of 67,000 and 72,000 that we have designated small MAG (S-MAG) and large MAG (L-MAG), respectively. The complete amino acid sequence of L-MAG and a partial amino acid sequence of S-MAG have been deduced from the nucleotide sequences of corresponding cDNA clones isolated from a lambda gt11 rat brain expression library. Based on their amino acid sequences, we predict that both proteins have an identical membrane spanning segment and a large extracellular domain. The putative extracellular region contains an Arg-Gly-Asp sequence that may be involved in the interaction of these proteins with the axon. The extracellular portion of L-MAG also contains five segments of internal homology that resemble immunoglobulin domains, and are strikingly homologous to similar domains of the neural cell adhesion molecule and other members of the immunoglobulin gene superfamily. In addition, the two MAG proteins differ in the extent of their cytoplasmically disposed segments and appear to be the products of alternatively spliced mRNAs. Of considerable interest is the finding that the cytoplasmic domain of L-MAG, but not of S-MAG, contains an amino acid sequence that resembles the autophosphorylation site of the epidermal growth factor receptor.     

The expression of recombinant large myelin-associated glycoprotein cytoplasmic domain and the purification of native myelin-associated glycoprotein from rat brain and peripheral nerve

The myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily existing as two isoforms (L-MAG and S-MAG) that are differentially expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively. The polypeptide chains of the two isoforms differ only by the carboxy terminus of their respective cytoplasmic domains, which most probably determine the isoform-specific functions. Here, we describe the expression of the L-MAG cytoplasmic domain as a GST fusion protein. The recombinant protein was used to raise polyclonal antibodies against the L-MAG-specific carboxy terminus and against the region of the MAG cytoplasmic domain common to both S-MAG and L-MAG. These antibodies, which function in dot blotting, Western blotting, and immunoprecipitation, were used to immunopurify native MAG from both rat brain and peripheral nerves in quantities and purity sufficient for the realization of most biochemical and functional studies. The antibodies and the recombinant and native MAG proteins provide much needed tools for the study of the common and isoform-specific properties and functions of L-MAG and S-MAG.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Protein Kinase FA/Glycogen Synthase Kinase-3 Predominantly Phosphorylates the In Vivo Site Thr97-Pro in Brain Myelin Basic Protein: Evidence for Thr-Pro and Ser-Arg-X-X-Ser as Consensus Sequence Motifs

Abstract: In a previous study, protein kinase F_A/glycogen synthase kinase-3 ( F_A/GSK-3 ) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F_A/GSk-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F_A/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr⁹⁷-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase F_A/GSK-3, implicating a physiologically relevant role of F_A/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT⁹⁴(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [³²P]MBP phosphorylated by kinase F_A/GSK-3, further indicating that kinase F_A/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.     

The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.

Phosphorylation of the myelin-associated glycoprotein (MAG) in the peripheral nervous system is demonstrated by immunoprecipitation from myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Phosphoamino acid analysis of immunoprecipitated MAG revealed the presence of radioactivity in phosphoserine, but not in phosphothreonine or phosphotyrosine. Only the shorter isoform of MAG (S-MAG) was detected by immunostaining of nitrocellulose sheets with anti-MAG anti-serum after enzymatic deglycosylation of immunoprecipitated MAG labeled in nerve slices. Autoradiography of the same Western blots revealed that most of the radioactive phosphate was in S-MAG, demonstrating that the polypeptide backbone of S-MAG is phosphorylated in the PNS.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

Mutual stimulation by phosphatidylinositol-4-phosphate and myelin basic protein of their phosphorylation by the kinases solubilized from rat brain myelin

Myelin basic protein and phosphatidylinositol-4-phosphate are phosphorylated in vitro by ATP and solubilized rat brain myelin. When both substrates are present together, the rate of phosphorylation of each is increased about eight-fold. It appears likely that the phosphate turnover of myelin basic protein and of phosphatidylinositol-4-phosphate are coupled in vivo.     

Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.     

Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [(33)P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylated in vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn(2+) over Mg(2+), can be inhibited by micromolar concentrations of Zn(2+) and Cd(2+), and is not Ca(2+)-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.     

In vivo phosphorylation of adaptors regulates their interaction with clathrin

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self- assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly.