Structural and functional aspects of phosphorylation of human fibronectins
Upon cathepsin D digestion of /sup 32/P-orthophosphate labeled fibronectin, two major phosphopeptides of molecular weight 160,000 and 140,000 were detected by SDS-PAGE and they gave rise upon reduction, to three phosphopeptides of molecular weight 85,000, 75,000 and 65,000. These peptides had little affinity for gelatin, but showed strong binding to heparin. This affinity was insensitive to divalent calcium ions, indicating that heparin binding was towards the COOH domain of the molecule. Limited thermolysin digestion of the protein generated five major phosphopeptides of molecular weight of 180,000, 43,000, 38,000, 26,000 and 5000 upon reduction. They migrated to similar positions under non-reducing conditions except that the 38,000 dalton fragment migrated to a 75,000 dalton position. These fragments did not adhere to gelatin, heparin or Staphylococcus aureus ligands. CNBr cleavage of /sup 35/S-methionine labeled fibronectin generated approximately 22 fragments while CNBr cleaveage of /sup 32/P-orthophosphate labeled fibronectin yielded one major phosphopeptide. Based upon the composite data, it is proposed that the major phosphorylation site is located on domain 7. The primary structure of the CNBr-phosphopeptide was determined by the H/sub 2/N-Pro-Leu-Asp-Val-Gln-Ala-Asp-Arg-Glu-Asp-Pse-Arg-Glu-COOH. In order to elucidate the nature of the presumptive kinase involved, an in vitro cell culture system was utilized. Judging by the amino acid sequence adjacent to the phosphorylation site of fibronectin, the substrate specificities of kinases and dependency of the phosphorylation on ligand(s), it is concluded that casein kinase I probably mediate the phosphorylation of fibronectin.
- Research Organization:
- Connecticut Univ., Storrs (USA)
- OSTI ID:
- 6902145
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACID SEQUENCE
AMINO ACIDS
ANTICOAGULANTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CARBOXYLIC ACIDS
CASEIN
CELL CULTURES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DIGESTION
DRUGS
ENZYMES
EVEN-ODD NUCLEI
HEMATOLOGIC AGENTS
HEPARIN
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
METHIONINE
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
MUCOPOLYSACCHARIDES
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
POLYPEPTIDES
POLYSACCHARIDES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SACCHARIDES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR 35
SULFUR ISOTOPES
TRACER TECHNIQUES
TRANSFERASES