We describe here an important function of the novel calmodulin kinase I isoform, Pregnancy Up-regulated Non-ubiquitous Calmodulin Kinase (Pnck). Pnck (also known as CaM kinase I₂) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared to benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly up-regulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly up-regulated in confluent and serum starved cells, compared to actively growing proliferating cells (Gardner, H. P., Seung, H. I., Reynolds, C., and Chodosh, L. A. (2000) Cancer Res. 60, 5571-5577). In spite of these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly down-regulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells over expressing Pnck. MAP kinase activation was strongly inhibited by EGFR-downregulation in the Pnck-overexpressing cells. The EGFR down regulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knock down of endogenous Pnck mRNA levels by siRNA transfection in human breast cancer cells resulted in up-regulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR down-regulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and may represent an attractive therapeutic target in EGFR-regulated oncogenesis.