Cuttlefish sperm protamines. 1. Amino acid sequences of two distinct variants

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.     

Primary structure of the chromosomal proteins MC1a, MC1b, and MC1c from the archaebacterium Methanothrix soehngenii

The chromosomal protein MC1 of Methanothrix soehngenii is a family of three variants a, b, and c. These are small basic polypeptides of 89, 87, and 90 residues, respectively. Their primary structures have been determined from automated sequence analyses of the intact proteins and from structural data provided by peptides derived from the variants by cleavage at aspartic acid, glutamic acid, arginine, and methionine residues. By comparison with variant b taken as reference, variants a and c present 18 and 24 differences, respectively. The extent of sequence homologies between protein MC1 from M. soehngenii and proteins MC1 from two other species of Methanosarcinaceae is only 60%. The sequences 17-35 and 45-58 of the protein MC1 appear well conserved. Deletions are observed in region 36-44. Many changes, most of them nonconservative, occur in the carboxyl-terminal third of the protein. However, proline residues at positions 68, 72, 76, and 82 remain strictly conserved. Predictive methods for secondary structures indicate a low content of alpha helix and beta sheet structures in proteins MC1.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases

The Gab family of docking proteins is phosphorylated in response to various growth factors and cytokines and serves to recruit multiple signaling proteins. Gab1 acts downstream from the Met-hepatocyte growth factor receptor, and Gab1 overexpression promotes Met-dependent morphogenesis of epithelial cells. Recruitment of Gab1 to Met or epidermal growth factor (EGF) receptors requires a receptor-binding site for the Grb2 adapter protein and a proline-rich domain in Gab1, defined as the Met-binding domain. To determine the requirement for Grb2 in Gab1 recruitment, we have mapped two Grb2 carboxyl-terminal SH3 domain binding sites conserved in Gab1 and related protein Gab2. One corresponds to a canonical Grb2-binding motif, whereas the second, located within the Gab1 Met-binding domain, requires the proline and arginine residues of an atypical PXXXR motif. The PXXXR motif is required but not sufficient for Grb2 binding, whereas an extended motif, PX3RX2KPX7PLD, conserved in Gab proteins as well as the Grb2/Gads-docking protein, Slp-76, efficiently competes binding of Grb2 or Gads adapter proteins. The association of Gab1 with Grb2 is required for Gab1 recruitment to the EGF receptor but not the Met receptor. Hence different mechanisms of Gab1 recruitment may reflect the distinct biological functions for Gab1 downstream from the EGF and Met receptors.     

Sp1-mediated transcriptional activation is repressed by Sp3.

Sp1, Sp3 (SPR-2) and Sp4 (SPR-1) are human sequence-specific DNA binding proteins with very similar structural features. In this report, we have analyzed Sp3 in direct comparison with Sp1. We have raised antibodies against both Sp1 and Sp3, and show that Sp3 protein, like Sp1, is expressed in various cell lines. Co-transfection experiments in different mammalian cell lines reveal that in contrast to Sp1 and Sp4, Sp3 is not able to activate several Sp1 responsive promoters. In addition, Sp3 also fails to activate reporter constructs in Drosophila SL2 cells lacking endogenous Sp factors. Instead, we find that Sp3 represses Sp1-mediated activation in a linear dose-dependent manner. A mutant of Sp3 lacking the DNA binding domain does not affect activation by Sp1, suggesting that the inhibition is most likely due to the competition with Sp1 for their common binding sites. To determine if any structurally similar domain of Sp3 is able to replace partially homologous domains of Sp1, we have generated chimeric proteins and tested their activation characteristics in gene transfer experiments. It appears that neither the glutamine-rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3. Our results suggest that Sp3 is an inhibitory member of the Sp family.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

Structural variants of human T200 glycoprotein (leukocyte-common antigen).

Structural variation in the primary structure of human T200 glycoprotein has been detected. Three cDNA variants have been characterized each of which encode T200 molecules that differ in size as a result of sequence differences in their amino-terminal regions. The largest form of the molecule is distinguished from the smallest by an insert of 161 amino acids, after the first eight amino-terminal residues. The other variant has an insert at the same location of 47 amino acids identical to residues 75-121 in the larger insert. Both extra domains are rich in serine and threonine residues and are likely to display multiple O-linked oligosaccharides. These structural variants which probably arise by cell-type-specific alternative splicing provide a molecular basis for the previously observed structural and antigenic heterogeneity of T200 glycoprotein. In addition to the variable amino-terminal region, the external domain of human T200 glycoprotein consists of a second cysteine-rich region of about 400 amino acids, a single transmembrane-spanning region and a large cytoplasmic domain of 707 amino acids shared by all of the structural variants and highly conserved between species. The gene encoding human T200 is located on the long arm of chromosome 1.     

Synthesis and DNA-binding ability of Sp1 protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Sp1-ZF2), its mutant (Sp1-ZF2/HT. E²⁰ → H, R²³ → T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoC1₂ indicated that the formation of zinc finger structure was affected not only by the hydrophobic amino acids but also by the change of the distance between Cys and His. Gel-retardat ion electrophoresis assays indicated that the Glu and Arg residues are very important for recognition. A single zinc finger like Sp1-ZF2 is able to bind DNA sequence specifically.     

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

The adaptor protein Gads (Grb2-related adaptor downstream of Shc) is implicated in coupling hemopoietic progenitor kinase-1 to the activated TCR

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.