cGMP-dependent Protein Kinase Iβ Regulates Breast Cancer Cell Migration and Invasion via a Novel Interaction with the Actin/Myosin-associated Protein Caldesmon

Abstract

SummaryThe two isoforms of type I cGMP-dependent protein kinase (PKGIa and PKGIb) differ in their first ,100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein-protein interactions. Using the PKGIb dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGIb-specific binding protein. PKGIb phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGIb and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGIb enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGIb in human breast cancer cells, suggesting that PKGIb is a potential target for breast cancer treatment.

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression

“…SP also enhanced cGMP levels in MCF-7 and MDA-231 tumor xenografts (Figure 1B). Both MCF-7 and MDA-231 cells express cGMP-dependent PK G-1β (24, 25). To measure its enzymatic activity we monitored phosphorylation of Ser 239 of the endogenous PKG substrate, VASP.…”

cGMP-dependent protein kinase Iβ phosphorylates and regulates the function of the actin/myosin-associatedprotein caldesmon

“…Using wild-type and mutant PKGIβ D/D domains as affinity probes in a proteomic screen and we identified the actin/myosin associated protein caldesmon as a PKGIβ specific interacting protein [2]. Using immunofluorescent staining, we found that PKGIβ and CaD colocalized with F-actin at lammellipodial structures at the edge of MDA-MB-231 cells.…”

Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co‐culture models of cells

“…Elevated expression of this protein was also observed in oral SCCs (Méndez et al., ) and its overexpression was associated with lymph node metastasis and poor prognosis in patients with oral cavity SCC (Chang et al., ). Additionally, it was shown that CALD1 phosphorylation reduced its binding to actin and myosin thus promoting pro‐migratory and pro‐invasive activities in breast cancer cells (Schwappacher et al., ). Hence, we hypothesised that de‐phosphorylation of CALD1 in HFs co‐cultured with HKs and FaDu cells or in SCCFs co‐cultured with FaDu cells shown in this study may significantly contribute to contractile activity, promote formation of actin stress fibres and reduce cell motility during transition of fibroblast to MFs or CAFs.…”