Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases implicated in the regulation of cell division and cell morphology. This subfamily includes the kinases LATS, Orb6, Cot-1, and Dbf2. We show here that Ndr is potently activated when intact cells are treated with okadaic acid, suggesting that Ndr is normally held in a state of low activity by protein phosphatase 2A. We mapped the regulatory phosphorylation sites of Ndr protein kinase and found that active Ndr is phosphorylated on Ser-281 and Thr-444. Mutation of either site to alanine strongly reduced both basal and okadaic acid-stimulated Ndr activity, while combined mutation abolished Ndr activity completely. Importantly, each of these sites (and also the surrounding sequences) are conserved in the kinase relatives of Ndr, suggesting a general mechanism of activation for kinases of this subfamily. Ser-281 and Thr-444 are also similar to the regulatory phosphorylation sites in several targets of the phosphoinositide-dependent protein kinase PDK1.1 However, PDK1 does not appear to function as an upstream kinase for Ndr. Thus, Ndr and its close relatives may operate in a novel signaling pathway downstream of an as-yet-unidentified kinase with specificity similar to, but distinct from, PDK1.Human Ndr is a nuclear serine/threonine protein kinase that has been highly conserved during evolution and which is expressed in almost all cell types of the body (1). Sequence comparisons show that, within the protein kinase superfamily, Ndr is most closely related to a subgroup of kinases known to be important in the control of cell growth, cell division, and cell morphology. This subgroup is exemplified by the kinases LATS, Orb6, Cot-1 (50 -60% catalytic domain identity with Ndr) and Dbf2 (40% catalytic domain identity with Ndr). Several indications suggest that kinases of this type function, either directly or indirectly, as negative regulators of cyclin/ cyclin-dependent kinase complexes. The mammalian LATS kinase (also called Wts) is the product of a tumor suppressor gene and possesses an NH 2 -terminal domain that is able to directly interact with and inhibit Cdc2 (2, 3). Consistent with this, LATS interacts genetically with Cdc2 in Drosophila. Orb6 overexpression delays the onset of mitosis in fission yeast, and this effect is dependent upon the presence of a functional Wee1 protein, implying that Orb6 can signal through Wee1 to downregulate Cdc2 activity (4). Dbf2 is part of a network of genes required for down-regulation of Cdc28-cyclin B at the end of mitosis in the budding yeast S. cerevisiae (5). Dbf2 is transiently activated at the metaphase/anaphase transition, coincident with changes in its phosphorylation status, and mutation of Dbf2 causes cells to arrest in late mitosis with high Cdc28 activity (6). Finally, the Cot-1 protein of Neurospora crassa is required for hyphal elongation, although it is not known how Cot-1 functions in this process (7). Ndr thus belongs to a subfamily of kinases that are important in cell growth con...
