Lipocortin 2 (annexin 2) is a major substrate for constitutive tyrosine kinase activity in chondrocytes.
Treatment of cultured bovine articular chondrocytes with 100 microM orthovanadate, in the absence of serum, results in the production of a single major tyrosine phosphorylated protein with an apparent molecular mass of 36 kDa (p36). Chondrocytes were found to contain proteins reactive with anti-lipocortin 1, 2, and 5 antibodies. p36 comigrated on SDS-polyacrylamide gels with lipocortin 2, but not with other members of the lipocortin family. The distribution of p36 between the particulate and soluble cell fractions was also similar to that of lipocortin 2. p36 that was purified on an anti-phosphotyrosine immunoaffinity column cross-reacted with anti-lipocortin 2 antibodies. Similarly, lipocortin 2 purified on an anti-lipocortin 2 immunoaffinity column reacted with anti-phosphotyrosine antibodies. Furthermore, cyanogen bromide cleavage fragments of purified lipocortin 2 and p36 were similar. These data demonstrate that the major constitutively tyrosine phosphorylated protein, in chondrocytes, is lipocortin 2. Tyrosine phosphorylated p36 required SDS buffers for extraction due to a loss of the tyrosine phosphate group under other solubilization conditions using Triton X-100 or sodium cholate. This study provides a system for the study of the effects of tyrosine phosphorylation on lipocortin 2 function. What role lipocortin 2 plays in chondrocyte biology remains to be determined.
Grima
D T
DT
Connective Tissue Research Group, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada.
Kandel
R A
RA
Pepinsky
B
B
Cruz
T F
TF
D000818:Animals; D017306:Annexin A2 / Q000302:isolation & purification / Q000378:metabolism*; D002358:Cartilage, Articular / Q000201:enzymology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D017364:Collagenases / Q000096:biosynthesis / Q000378:metabolism*; D015870:Gene Expression; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D017306:Annexin A2; D010750:Phosphoproteins; D012333:RNA, Messenger; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017364:Collagenases