Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.     

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.     

Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins

Glycoproteins which contain about 45 mol% proline were dramatically induced in mouse parotid and submandibular glands by isoproterenol treatment, but these unusual proteins were not detected in control animals. These acid-soluble substances were obtained by extracting tissues with 10% trichloroacetic acid, as reported previously for isolating proline-rich proteins from rat submandibular glands (Mehansho, H., and Carlson, D.M. (1983) J. Biol. Chem. 258, 6616-6620). Three major proline-rich glycoproteins were induced in parotid glands with apparent molecular weights of 66,000 (GP-66p), 45,000 (GP-45p), and 27,000 (GP-27p), whereas only one such protein was expressed by the submandibular glands (66,000 (GP-66sm]. Both GP-66p and GP-66sm contained about 19% carbohydrate with the following molar ratios, respectively; GalNAc, 1.0, 1.0; Gal, 1.6, 2.3; GlcNAc, 0.8, 1.1; sialic acid, 0.9, 1.9. The peptide chains of GP-66p and GP-66sm appear to be identical by amino acid compositions, glycopeptide analysis, and preliminary amino acid sequencing data. Northern blot analysis of RNAs from parotid glands of normal and isoproterenol-treated rats, probed with a 32P-labeled proline-rich protein cDNA, confirmed that control animals were devoid of mRNAs encoding these proteins and that isoproterenol treatment dramatically induced expression of these genes. Feeding sorghum high in tannins caused changes in the parotid glands similar to those observed upon isoproterenol treatment, as noted earlier with rats (Mehansho, H., Hagerman, A., Clements, S., Butler, L., Rogler, J., and Carlson, D.M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3948-3952). These glycoproteins have high affinities for tannins as demonstrated by competitive binding curves.     

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.     

The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.     

Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.     

Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development.

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-D-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C. , Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569-582; Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583-592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile alpha motif domain, and a proline-rich region. By characterizing Shank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120-240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.     

Basic proline-rich proteins from human parotid saliva: complete covalent structures of proteins IB-1 and IB-6

The complete amino acid sequences of two basic proline-rich proteins, IB-1 and IB-6, from human parotid saliva have been determined. Fragments for sequence analysis were obtained by enzymatic digestions. The proteins have molecular weights of 9571 (IB-1) and 11,530 (IB-6) and contain 34 and 39 mol % proline, respectively. IB-1 and IB-6 contain an identical sequence of 54 residues except for an alanine in position 52 of IB-6, where IB-1 has proline. An unusually high number of repeated sequences occurs in both molecules. IB-1 has a blocked amino-terminal residue, pyroglutamic acid, and also contains one phosphoserine residue in position 8. The relationship of these proteins to the basic proline-rich protein IB-9 [Kauffman, D., Wong, R., Bennick, A., & Keller, P. (1982) Biochemistry 21, 6558-6562] and to other salivary proline-rich proteins is discussed.     

Isolation of early viral proteins from poxvirus-infected chick embryo fibroblasts by DNA-cellulose chromatography and inhibition of their synthesis by chicken interferon

Up to seven early poxvirus-specific proteins have been isolated from vaccinia-WR-infected and cowpox-virus-infected chick embryo fibroblasts by affinity chromatography on native DNA-cellulose columns. The proteins have been characterized by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by nonequilibrium pH-gradient electrophoresis. The molecular weights of the viral proteins were determined by comparison with proteins of known molecular weight and are comparable to several of the vaccinia-WR-specific DNA-binding proteins isolated previously from infected L-929 cells by Solosky J. M., Esteban M. and Holowczak J.A. [J. Virol. 25, 263-273 (1978)]. The viral proteins binding reversibly to native DNA have been classified as immediate early viral gene products. Synthesis of cowpox-virus-induced early DNA-binding proteins is inhibited in chick cells pretreated with homologous interferon at a concentration of 500--1000 units/ml.     

Characterization and sequence analysis of a developmentally regulated putative cell wall protein gene isolated from soybean

A cDNA clone, pTU04, which hybridizes to two different sizes of mRNA on Northern blots was isolated from soybean suspension culture cell poly(A) RNA. Northern analysis reveals that meristematic tissue produces a 1050-nucleotide mRNA while quiescent mature cells produce primarily a 1220-nucleotide mRNA homologous to pTU04. The cDNA and its corresponding genomic clone have been partially characterized. The nucleotide sequence of the gene predicts a proline-rich protein, designated SbPRP1, which contains a signal peptide sequence and 43 repeats of a sequence consisting primarily of Pro-Pro-Val-Tyr-Lys (CCA-CCA-GTT-TAC-AAG). From nuclease S1 and hybrid-select translation analyses, the cDNA clone pTU04 appears to represent the mRNA for the mature tissue 1220-nucleotide RNA observed on Northern blots. Although there is no direct proof that the encoded protein is a cell wall protein, it has the properties similar to previously isolated cell wall proteins: 1) it is very basic with a high content of Pro, Tyr, and Lys; 2) it has similar hydropathic properties; and 3) its repeating unit shares sequence homology with that of more highly characterized cell wall proteins, generally termed extensin (Chen, J., and Varner, J. E. (1985) EMBO J. 4, 2145-2151; Smith, J. J., Muldoon, E. P., Willard, J. J., and Lamport, D. T. A. (1986) Phytochemistry 25, 1021-1030.     

Membrane binding characteristics of two forms of transforming growth factor-beta

Cartilage-inducing factors A and B (CIF-A and CIF-B) from bovine bone have recently been identified as transforming growth factor-beta (TGF-beta) (Seyedin, S.M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem., 261, 5693-5695) and a unique protein homologous to TGF-beta (Seyedin S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987) J. Biol. Chem., 262, 1946-1949), respectively. Although the biological activities of TGF-beta and CIF-B are similar, the divergence of CIF-B from the highly conserved amino acid sequence of TGF-beta prompted an investigation of its receptor binding properties. Three classes of cell surface binding components were identified. Class A has exclusive affinity for TGF-beta; class B has greater affinity for CIF-B; and class C has equal affinity for both proteins. A high molecular weight component, the predominant binding species, was further characterized and shown to consist of two components that are either class B or class C. The differential binding properties of TGF-beta and CIF-B to cell surface components suggest that there are biological activities unique to each of the proteins.     

Quaternary structure of rice nonsymbiotic hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.     

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.     

Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.     

NH2-terminal acetylation of ribosomal proteins of Saccharomyces cerevisiae.

Using a mutant of Saccharomyces cerevisiae defective in the NAT1 gene, that encodes one of the NH2-terminal acetyltransferases, we have identified 14 ribosomal proteins whose electrophoretic mobility at pH 5.0 suggests they carry an additional charge, presumably due to the lack of NH2-terminal acetylation. At least 30 other ribosomal proteins from the mutant are electrophoretically normal. Attempted NH2-terminal analysis of most of the presumed acetylated proteins from wild type cells indicated that all were blocked. NH2-terminal analysis of the same proteins from the nat1 mutant strain yielded unique sequences. Each one carries an NH2-terminal serine. We conclude that these are normally acetylated due to the presence of the NAT1 gene product. It seems surprising that cells whose ribosomes have been altered to this degree grow rather well and synthesize the same spectrum of proteins as do wild type cells (Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S. Gribskov, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075). Finally, this analysis has provided the first sequence information available for several of the acetylated ribosomal proteins and for one non-acetylated ribosomal protein, which is clearly the product of the MFT1 gene (Garrett, J. M., Singh, K. K., Vonder Haar, R. A., and Emr. S. D. (1991) Mol. Gen. Gen. 225, 483-491).     

Biosynthesis of salivary proteins in the parotid gland of the subhuman primate, Macaca fascicularis. Cell-free translation of the mRNA for a proline-rich glycoprotein and partial amino acid sequence and processing of its signal peptide

The major anionic proline-rich proteins in the parotid and submandibular secretions of subhuman primates and man perform the important biological function of inhibiting crystal growth of calcium phosphate salts from saliva, which is supersaturated with calcium phosphate salts, thereby preventing excess deposition of hydroxylapatite on tooth surfaces. The present work was initiated as a first step towards investigating proline-rich protein biosynthesis in parotid glands using the subhuman primate, Macaca fascicularis, as a model system. RNA was isolated from macaque parotid glands and separated into poly(A)-enriched and poly(A)-deficient fractions by chromatography on oligo(dT)-cellulose. The mRNAs in both fractions promoted incorporation of radiolabeled amino acids into polypeptides in an mRNA-dependent reticulocyte lysate translation system. Five major proline-rich polypeptides were detected and one of these was shown to be the in vitro precursor of the major anionic macaque proline-rich protein (MPRP), which is the structural and functional counterpart of the major anionic proline-rich proteins in the parotid and submandibular secretions of man (Oppenheim, F.G., Offner, G.D., and Troxler, R.F. (1982) J. Biol. Chem. 257, 9271-9282). Radiosequencing of the material in anti-MPRP immune precipitates showed that the in vitro precursor of MPRP contained an 18-residue signal peptide. The in vitro precursor of MPRP was processed in dog pancreas vesicles to a form with a lower apparent Mr and with an NH2-terminal amino acid sequence identical to that of native MPRP. The phenylthiohydantoin derivatives of Ala and Ile were detected at residue 9 and those of Val and Met were detected at residue 16 of the signal peptide. This indicated that the in vitro precursor of MPRP, which migrated electrophoretically as a single band in anti-MPRP immune precipitates, contained two different in vitro polypeptides derived from two different mRNAs. These results are discussed in the context of the genetic polymorphism among the major anionic proline-rich proteins in the parotid and submandibular secretions of man.     

Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region

F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCF(Fbx7) complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes.