Mechanism of Ca2+-mediated regulation of NDR protein kinase through autophosphorylation and phosphorylation by an upstream kinase.
NDR1 (nuclear Dbf2-related) is a serine/threonine protein kinase belonging to subfamily of kinases implicated in the regulation of cell division and morphology. Previously, we demonstrated that the activity of NDR1 is controlled by phosphorylation of two regulatory residues, Ser-281 and Thr-444. Moreover, we found that NDR1 becomes activated through a direct interaction with EF-hand Ca(2+)-binding proteins of the S100 family. In this work, we characterize this regulatory mechanism in detail. We found that NDR1 autophosphorylates in vitro predominantly on Ser-281 and to a lesser extent on Thr-74 and Thr-444. All of these residues proved to be crucial also for NDR1 activity in vivo; however, in contrast to Ser-281 and Thr-444, Thr-74 seems to be involved only in binding to S100B rather than directly regulating NDR1 activity per se. When we added Ca(2+)/S100B, we observed an increased autophosphorylation on Ser-281 and Thr-444, resulting in stimulation of NDR1 activity in vitro. Using phosphospecific antibodies, we found that Ser-281 also becomes autophosphorylated in vivo, whereas Thr-444 is targeted predominantly by an as yet unidentified upstream kinase. Significantly, the Ca(2+)-chelating agent BAPTA-AM suppressed the activity and phosphorylation of NDR1 on both Ser-281 and Thr-444, and specifically, these effects were reversed when we added the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase pump inhibitor thapsigargin.
Tamaskovic
Rastislav
R
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel CH-4058, Switzerland.
Bichsel
Samuel J
SJ
Rogniaux
Helene
H
Stegert
Mario R
MR
Hemmings
Brian A
BA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D029721:Drosophila Proteins; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D029721:Drosophila Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; C419769:trc protein, Drosophila; D017346:Protein Serine-Threonine Kinases; D002118:Calcium