Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin.

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.

Carboxyl methylation of deamidated calmodulin increases its stability in Xenopus oocyte cytoplasm. Implications for protein repair.

The widely distributed protein-L-isoaspartate(D-aspartate) O-methyltransferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repair or metabolism of damaged cellular proteins containing L-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT-catalyzed methylation on the stability of isoaspartyl-containing proteins in cells, Xenopus laevis oocytes were microinjected with both deamidated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blotting of oocyte extracts. The experiments indicate that deamidated HA-CaM is degraded after microinjection, while nondeamidated HA-CaM is stable. Kinetic analysis is consistent with the entry of microinjected HA-CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA-CaM bound to the heterogeneous pool of oocyte CaM binding proteins detected by an overlay procedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibition of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM degradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins.

Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts.

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

The catalytic subunit of the cAMP-dependent protein kinase of ovine sperm flagella has a unique amino-terminal sequence.

The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated. Ram sperm C was purified and found by mass spectrometry (MS) to be approximately 890 Da smaller than Calpha, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Calpha, but differed from the predicted sequences for the Cbeta and Cgamma isoforms. MS analysis of 2-nitro-5-thiocyanatobenzoic acid fragments showed that the mass difference originated in the amino-terminal region. A unique blocked amino-terminal fragment was isolated from sperm C and sequenced by a combination of tandem mass spectrometry and Edman degradation of a subfragment. The results revealed that the amino-terminal myristate and the first 14 amino acids of Calpha are replaced by an amino-terminal acetate and six different amino acids in sperm C. The predicted mass difference due to these changes is 899 Da. The region of homology between sperm C and Calpha begins at the exon 1/exon 2 boundary in Calpha, suggesting that sperm C results from use of an alternate exon 1 in the Calpha gene. The different amino terminus of sperm C may be related to a unique requirement for localization of the "free" C subunit within the sperm flagellum.

The alpha-dystroglycan-beta-dystroglycan complex. Membrane organization and relationship to an agrin receptor.

Aberrant expression of the dystrophin-associated protein complex is thought to underlie the pathogenesis of Duchenne dystrophy, Becker muscular dystrophy, and severe childhood autosomal recessive muscular dystrophy. Recently, our laboratory identified an agrin receptor from Torpedo electric organ postsynaptic membranes. It is a heteromer of 190- and 50-kDa subunits with similarity to two components of the dystrophin-associated protein complex of alpha- and beta-dystroglycan. We now confirm the relationship between the Torpedo agrin receptor and mammalian dystroglycans and provide further information about the structure of the alpha-dystroglycan-beta-dystroglycan complex. The sequences of three peptides from each Torpedo subunit were 69% identical to mammalian dystroglycans. An antiserum to mammalian beta-dystroglycan recognizes the Torpedo 50-kDa polypeptide. Additionally, like alpha-dystroglycan, the 190-kDa agrin receptor subunit binds laminin. Previous studies have indicated that alpha- and beta-dystroglycan arise by cleavage of a precursor protein. Tryptic peptide mapping of both subunits and amino-terminal sequencing of Torpedo beta-dystroglycan indicate a single cleavage site, corresponding to serine 654 of the mammalian dystroglycan precursor. Gel electrophoresis analysis indicates there is at least one intrachain disulfide bond in beta-dystroglycan. These results provide precise primary structures for alpha- and beta-dystroglycan.

Ricin A chain can be chemically cross-linked to the mammalian ribosomal proteins L9 and L10e.

Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between 125I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.

Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans.

The selective concentration of neurotransmitter receptors at the postsynaptic membrane is an essential aspect of synaptic differentiation and function. Agrin is an extracellular matrix protein that is likely to direct the accumulation of acetylcholine receptors and several other postsynaptic elements at developing and regenerating neuromuscular junctions. How agrin interacts with the membrane to bring about these changes is unknown. We now report the identification and purification of a protein complex from Torpedo electric organ postsynaptic membranes that is likely to serve as an agrin receptor. The native receptor is a heteromeric complex of two membrane glycoproteins of 190 kDa and 50 kDa. The 190 kDa subunit is sufficient to bind ligand. Peptide sequence analysis revealed that the 190 kDa and 50 kDa subunits are related to the dystrophin-associated glycoproteins alpha- and beta-dystroglycan, respectively. No other candidate agrin receptors were detected. The identification of the agrin receptor opens new avenues toward a mechanistic understanding of synapse differentiation.

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.