Related Experiment Videos
Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus
B K Brott1, B A Pinsky, R L Erikson
1Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA. bbrott@fas.harvard.edu
Abstract:
Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase, Nlk, that is a murine homolog of the Drosophila nemo (nmo) gene. The Nlk amino acid sequence is 54. 5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4. 0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60-70% of Nlk is localized to the nucleus, whereas 30-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.
Insights
We identified Nlk, a novel kinase similar to Erk/MAPKs and Cdks, highly expressed in the adult mouse brain. Nlk
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are crucial regulators of cell cycle progression, growth, and apoptosis.
- Understanding novel kinases involved in these processes is vital for deciphering cellular regulation.
- The Drosophila nemo (nmo) gene plays a role in development, but its mammalian homologs and functions are less understood.
Purpose of the Study:
- To clone and characterize a murine homolog of the Drosophila nemo (nmo) gene, named Nlk.
- To investigate the structural, expression, and functional properties of the Nlk kinase.
- To determine the potential role of Nlk in cellular processes regulated by Erk/MAPKs and Cdks.
Main Methods:
- Cloning of the Nlk gene and sequence analysis to determine homology to known kinases.
- Expression analysis using Northern blotting in adult mouse tissues and during embryogenesis.
- Transient expression in COS7 cells to assess kinase activity, autophosphorylation, and effects of mutations.
- Subcellular localization studies using fractionation and immunofluorescence microscopy.
Main Results:
- Nlk shares significant sequence similarity with Erk-2 and Cdc2, suggesting a potential dual homology.
- Nlk exhibits high expression in adult mouse brain as a 4.0-kb transcript, with lower levels in other tissues.
- Mutations in the ATP-binding site or a putative activating threonine residue abolish Nlk kinase activity.
- Nlk is predominantly localized to the nucleus (60-70%), with a smaller fraction in the cytoplasm.
Conclusions:
- Nlk represents a novel mammalian kinase with structural homology to both Erk/MAPK and Cdk families.
- Its high expression in the brain suggests a specific role in this tissue.
- Nlk possesses intrinsic kinase activity that is dependent on key catalytic residues and shows predominantly nuclear localization.
- Nlk and Nmo may represent the founding members of a new kinase subfamily with unique regulatory functions.