Laminin-1 is phosphorylated by ecto-protein kinases of monocytes.
Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties. In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([gamma-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS-PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls. These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes.
Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece.
Varvara
V
Trachana
Efthymios
E
Christophorides
Kokkona
K
Kouzi-Koliakos
George
G
Koliakos
D000925:Anticoagulants / Q000494:pharmacology; D001485:Basement Membrane / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D026121:Indole Alkaloids; D007797:Laminin / Q000378:metabolism* / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*
D000925:Anticoagulants; D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007797:Laminin; C121346:laminin 1; D006493:Heparin; C049985:staurosporine aglycone; D011494:Protein Kinases; C034454:ectoprotein kinase; D047390:Casein Kinase II