The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B.
Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.
Gentil
B J
BJ
Département de Biologie Moléculaire et Structurale du Commissariat à l'Energie Atomic, INSERM EPI-0104 and Laboratoire de Chimie des Proteines DBMS-CP, CEN-G, 38054 Grenoble, France.
Delphin
C
C
Mbele
G O
GO
Deloulme
J C
JC
Ferro
M
M
Garin
J
J
Baudier
J
J
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000378:metabolism*
C075176:AHNAK protein, human; C489572:Ahnak protein, mouse; C489573:Ahnak protein, rat; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; C579159:S100b protein, mouse; C579158:S100b protein, rat; D015032:Zinc; D002118:Calcium