Abstract
Pregnancy upregulated non-ubiquitous calmodulin kinase (Pnck), a novel calmodulin kinase, is significantly overexpressed in breast and renal cancers. We present evidence that at high cell density, overexpression of Pnck in HEK 293 cells inhibits serum-induced extracellular signal-regulated kinase (ERK1/ERK2) activation. ERK1/2 inhibition is calcium-dependent and Pnck kinase activity is required for ERK1/2 inhibition, since expression of a kinase-dead (K44A) and a catalytic loop phosphorylation mutant (T171A) Pnck protein is unable to inhibit ERK 1/2 activity. Ras is constitutively active at high cell density, and Pnck does not alter Ras activation, suggesting that Pnck inhibition of ERK1/2 activity is independent of Ras activity. Pnck inhibition of serum-induced ERK1/2 activity is lost in cells in which phosphatase and tensin homolog (PTEN) is suppressed, suggesting that Pnck inhibition of ERK1/2 activity is mediated by PTEN. Overexpression of protein phosphatase-active but lipid phosphatase-dead PTEN protein inhibits ERK1/2 activity in control cells and enhances Pnck-mediated ERK1/2 inhibition, suggesting that Pnck increases availability of protein phosphatase active PTEN for ERK1/2 inhibition. Pnck is a stress-responsive kinase; however, serum-induced p38 MAP kinase activity is also downregulated by Pnck in a Pnck kinase- and PTEN-dependent manner, similar to ERK1/2 inhibition. Pnck overexpression increases proliferation, which is inhibited by PTEN knockdown, implying that PTEN acts as a paradoxical promoter of proliferation in ERK1/2 and p38 MAP kinase phosphorylation-inhibited, Pnck-overexpressing cells. Overall, these data reveal a novel function of Pnck in the regulation of ERK1/2 and p38 MAP kinase activity and cell proliferation, which is mediated by paradoxical PTEN functions. The possible biological implications of these data are discussed.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 961-973 |
| Number of pages | 13 |
| Journal | Cell Cycle |
| Volume | 13 |
| Issue number | 6 |
| DOIs | |
| State | Published - Mar 15 2014 |
| Externally published | Yes |
Bibliographical note
Funding Information:This work was supported by a US Department of Defense Concept award in breast cancer (BC103388, to T.B.D.), a grant from Susan G. Komen for the Cure (BCTR0707114, to T.B.D.), an American Cancer Society Institutional Research Grant (IRG-97-152-16, to T.B.D.), a Lombardi Comprehensive Cancer Center (LCCC) Nina Hyde Breast Cancer Research grant (to T.B.D.), and a Lombardi Comprehensive Cancer Center Support Grant Developmental Fund Award (CCSG DFA) (to T.B.D.). The views expressed in this publication are solely those of the authors with which the US Department of Defense and National Institutes of Health may not necessarily agree. The Raf-1 GST RBD 1–149 plasmid was obtained from Dr Channing J Der through the Addgene plasmid repository (plasmid number 13338). We sincerely acknowledge the help from LCCC Shared Resources, such as the Tissue Culture Shared Resource, Flow Cytometry and Cell Sorting Shared Resource, and Microscopy and Imaging Shared Resource, which are supported by a Cancer Center Support Grant P30-CA051008 from National Institutes of Health (NIH/NCI). Help from Dr Todd A Waldman and Dr JS Kim of the Lombardi Comprehensive Cancer Center in providing wt and mutated PTEN plasmids and PTEN shRNA is gratefully acknowledged.
Keywords
- Breast cancer
- Cell signaling
- ERK1/2
- PTEN
- Phosphorylation
- Phosphotyrosine
- Pnck
- Proliferation
- Protein phosphatase
- p38 MAPK
