Proteoglycan synthesis is increased in cells with impaired clathrin-dependent endocytosis.
Overexpression of a GTPase deficient dynamin mutant in HeLa dynK44A cells causes a block in clathrin-dependent endocytosis. When endocytosis is inhibited, these cells incorporate higher levels of [(35)S]sulfate into both cellular and secreted macromolecules and larger amounts of proteoglycans such as syndecan and perlecan are immunoprecipitated from [(35)S]sulfate-labelled lysates. Gel filtration and ion-exchange chromatography revealed that the increased [(35)S]sulfate incorporation into proteoglycans was not due to significant differences in size or density of negative charge of glycosaminoglycan chains attached to proteoglycan core proteins. On the other hand, measurements of the syndecan-1 mRNA level and of [(3)H]leucine-labelled perlecan after immunoprecipitation supported the idea that the increased [(35)S]sulfate incorporation into proteoglycans was due to a selective increase in the synthesis of proteoglycan core proteins. Interestingly, the activity of protein kinase C was increased in cells expressing mutant dynamin and inhibition of protein kinase C with BIM reduced the differences in [(35)S]sulfate incorporation between cells with normal and impaired clathrin-dependent endocytosis. Thus, the activation of protein kinase C observed upon inhibition of clathrin-dependent endocytosis may be responsible for the increased synthesis of proteoglycans.
Llorente
A
A
Department of Biochemistry, The Norwegian Radium Hospital, Montebello, Norway.
Prydz
K
K
Sprangers
M
M
Skretting
G
G
Kolset
S O
SO
Sandvig
K
K
D000818:Animals; D002460:Cell Line; D019765:Chondroitin ABC Lyase; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology*; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014316:Tritium
D002966:Clathrin; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014168:Transferrin; D014316:Tritium; D016220:Fibroblast Growth Factor 1; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D034281:Dynamins; D019765:Chondroitin ABC Lyase; D007930:Leucine