Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation

Abstract

We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Iβ2) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571–5577, 2000). Despite 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 downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis.

PTEN-mediated ERK1/2 inhibition and paradoxical cellular proliferation following Pnck overexpression

“…5 We showed that this phenomenon is dependent on cell density, with EGFR being protected from Pnck-induced degradation in cells grown at high cell densities. We were intrigued to discover, however, that Pnck-induced suppression of ERK1/2 activity is not relieved in cells grown at high cell density.…”

“…Under those conditions Pnck-induced EGFR degradation reduces EGF-induced EGFR tyrosine phosphorylation, which, in turn, is reflected in reduced Shc tyrosine phosphorylation. 5 EGFR tyrosine kinase activity is, however, required for ERK1/2 activity in both Pnck (wt and K44A)-expressing and Neo cells, since pre-incubation with AG1478, a specific EGFR kinase inhibitor, completely blocks EGFR tyrosine phosphorylation, Shc tyrosine phosphorylation, and ERK1/2 phosphorylation (Fig. S2).…”

“…Since Pnck is a member of calmodulin kinase family, and the activity these proteins is often regulated by calcium, we next examined whether Pnck requires calcium for its ERK1/2-suppressive activity. Cells were pre-incubated with BAPTA-AM, a calcium chelator, 5,8,9 before serum. As seen in Figure S1, pre-treatment with BAPTA-AM blocked the ability of Pnck to inhibit serum-induced ERK1/2 activity, suggesting that this activity of Pnck is calciumdependent.…”

“…(A) pnck inhibits serum-induced eRK1/2 activity. Cells transfected with Neo control (lane 1-2), HA-pnck (lane 3-4), t171A mutant (lane [5][6], and K444A mutant (lane 7-8) expression constructs were grown at and high cell density, serum starved overnight, and treated without or with 20% heat-inactivated serum for 10 min. equal amounts of total protein containing lysates were immunoblotted for phosphoeRK1/2 (t202/Y204) pAb (p-eRK1/2), eRK1/2, phospho-Akt (S473), Akt and HA-pnck expression.…”

“…3,4 The biological implications of the expression of Pnck in the pregnant mammary gland or its overexpression in breast and renal tumors are not, however, well understood. We have previously shown that enforced expression of Pnck results in the ligandindependent degradation of epidermal growth factor receptor (EGFR), 5 and recently reported that Hsp90 is differentially phosphorylated in Pnck-overexpressing cells, 6 which may cause the dissociation of EGFR from the Hsp90 chaperone complex and result in the subsequent protea-lysosomal degradation of EGFR. 6 While conducting this work, we observed that Pnckinduced EGFR degradation is cell density-dependent.…”