Susan A. Rotenberg

Scanning electrochemical microscopy of living cells Part 2. Imaging redox and acid:basic reactivities

Abstract Amperometric feedback and potentiometric modes of the scanning electrochemical microscope (SECM) have been used to image the topography and map redox and acid–base activities in single mammalian cells. The topographic images of cells were obtained using hydrophilic redox mediators, which cannot penetrate the cell membrane. In contrast, with a hydrophobic mediator one can map redox reactivity with a micrometer or submicrometer spatial resolution. The images obtained with oxygen used as a redox mediator show the distribution of the diffusion rate of oxygen in the cell membrane and inside the cell. The acid release by the cell was imaged with a Sb tip in a potentiometric mode, and the possibility of redox and pH imaging of the same cell with same tip is demonstrated. Significant differences were detected in the redox and pH images of normal human breast epithelial cells and metastatic breast cancer cells.

Immunohistochemical Analysis of Advanced Human Breast Carcinomas Reveals Downregulation of Protein Kinase Cα

Forty-six advanced-stage human breast carcinoma specimens were evaluated by immunohistochemistry for PKCα expression and compared with 25 samples of normal adjacent breast tissue. For normal tissue, the median staining of ductal epithelia was of moderate intensity. No staining was observed for 67% of tumor specimens, and only 4% showed intensities greater than the median observed in normal tissue. Faint to moderate PKCα staining was observed in the stroma, inflammatory cells, and fibroblasts of tumors but was absent in normal tissue. These findings demonstrate that downregulation of PKCα protein occurs in epithelial cells of advanced breast tumors (p<0.001).

Photoinduced inactivation of protein kinase C by dequalinium identifies the RACK-1-binding domain as a recognition site.

1,1′-Decamethylenebis-4-aminoquinaldinium diiodide (DECA; dequalinium) is an anti-tumor agent and protein kinase C (PKC) inhibitor whose mechanism of action with PKC is unknown. This study reports that with human PKCα, DECA exhibited competitive inhibition (K i = 11.5 ± 5 μm) with respect to RACK-1 (receptor for activatedC kinase-1), an adaptor protein that has been proposed to bind activated PKC following translocation (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180–24187). When exposed to UV light, DECA covalently modified and irreversibly inhibited PKC (α or β), with IC50 = 7–18 μm. UV/DECA treatment of synthetic peptides modeled after the RACK-1-binding site in the C2 region of PKCβ induced modification of Ser218-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr226, but not of a control peptide. This modification occurred at a tryptophan residue (Trp223) that is conserved in all conventional PKC isoforms. In overlay assays with native RACK-1 that had been immobilized on nitrocellulose, UV-treated control PKCα bound well to RACK-1, whereas UV/DECA-inactivated PKCα had reduced binding activity. The significance of these findings is shown with adenocarcinoma cells, which, when pretreated with 10 μmDECA and UV light, exhibited diminished 12-O-tetradecanoylphorbol-13-acetate-induced PKCα translocation. Overall, this work identifies DECA as a tool that prevents PKC translocation by inhibiting formation of the PKC·RACK-1 complex.

Protein Kinase C in Neoplastic Cells

Publisher Summary This chapter discusses protein kinase C (PKC) in neoplastic cells. PKC is a Ca2+ and phospholipid-dependent protein kinase that phosphorylates intracellular substrates on serine and threonine residues. Current models of PKC structure depict inactive PKC as having its N-terminal pseudo substrate segment tucked into the protein structure and in contact with the substrate binding site. This folded, inactive form undergoes a conformational change upon addition of PKC activators such that the enzyme auto-phosphorylates and physiological substrates are admitted to the binding site. In the context of signal transduction, intracellular activation of PKC is generally believed to occur via growth factor receptor-mediated activation of phospholipase C. In addition, the epidermal growth factor (EGF) receptor-kinase is phosphorylated at threonine-654 by PKC both in vitro and in vivo. The role of PKC in the biology of cancer has been studied in the context of its function as high-affinity receptor for the phorbol ester tumor promoters, and as an enzyme that mediates the action of certain growth factors and oncogenes. Preliminary studies demonstrate that human colon, breast, and brain tumors display altered levels of PKC when compared to their normal counterparts.

Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells

Background: Cdc42 effector protein-4 (CEP4) is a substrate of protein kinase C (PKC) in human breast cells. Results: Phosphorylation at two defined positions in CEP4 causes it to dissociate from Cdc42 and consequently to stimulate cell movement. Conclusion: CEP4 phosphorylation at Ser18 and Ser80 by PKC promotes cell movement. Significance: This work describes a novel PKC-stimulated signaling pathway by which human breast cells acquire metastatic potential. Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser18, Ser77, Ser80, and Ser86) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser18 and Ser80 were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser18 and Ser80 causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.

Abstract 57: Substrates of protein kinase C drive cell rac1-dependent motility

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA This laboratory has identified and/or characterized substrates of PKC that upon phosphorylation give rise to motility, an aspect of metastasis. By use of the traceable kinase method, we discovered that alpha-tubulin and Cdc42 effector protein-4 (CEP4) are PKC substrates. Phosphorylation of alpha-tubulin stimulates its incorporation into microtubules (MTs), consequently increasing the stability and prolonged growth of MTs and leading to the activation of the small GTPase Rac1. CEP4 undergoes phosphorylation by PKC that results in its release from Cdc42, whereupon CEP4 binds a guanine nucleotide exchange factor (GEF) that in turn activates Rac1 GTPase. These results imply that Rac1 acts as a node in pathways driven by phosphorylated PKC substrates. Since translocation of IQGAP to the membrane is known to be promoted by Rac1, a role is explored in non-transformed human MCF-10A cells that express a specific phospho-mimetic mutant substrate. In addition, the phospho-mimetic mutant for each substrate expressed in human metastatic MDA-MB-231 cells produces different morphologies in 3-D growth assays. This research is being supported by NIH CA125632. Citation Format: Susan A. Rotenberg, Xin Zhao, Shatarupa De. Substrates of protein kinase C drive cell rac1-dependent motility. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 57. doi:10.1158/1538-7445.AM2015-57

Abstract 5290: Motility of B16 mouse melanoma cells is driven by phospho-MARCKS

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Phosphorylation of Myristoylated Alanine-rich C-Kinase Substrate (MARCKS) by protein kinase Cα (PKCα) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that cytoplasmic phosphoMARCKS directly promotes motile behavior of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas weakly motile F1 cells express negligible levels. When treated with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibit a dramatic increase in phosphoMARCKS coincident with a 5-fold increase in motility. This motility is lost if OA-treated cells are also treated with a shRNA reagent targeted to MARCKS. Pre-treatment of cells with calphostin C (a PKC inhibitor) prevents OA-mediated elevation of phosphoMARCKS. These findings imply that PKC is highly active in F1 cells but that its phosphorylation of MARCKS is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility is established with a pseudo-phosphorylated mutant of GFP-MARCKS in which Asp residues replaced Ser residues known to be phosphorylated by PKCα. This mutant localizes to the cytoplasm and engenders three-fold higher motility in F1 cells. However, when the mutant is fused to a nuclear localization signal, motility is substantially decreased. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localizes to the cytoplasm, blocks motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These findings demonstrate the importance of phosphoMARCKS to the metastatic potential of melanoma cells, and reveal a previously undocumented cytoplasmic role for this phospho-protein. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5290.

Optical speckle angular correlation and fractal property of a composite of cancer/normal cell layers with application for laboratory monitoring of drug efficacy

Breast cancer cells and normal cells were grown on glass substrates and investigated via laser generated speckles. The optical speckle pattern of a layer was investigated via angular correlation and fractal dimension analysis. A porous silicate slab with various water contents was used as calibration. The angular correlation and its associated Fourier transform results were consistent with the property of the cells. The speckle intensity data can be treated as a random series and the Higuchi method was used to explore the fractal property of the random series. The fractal dimension results differentiated the cancer cells (fractal dimension about 1.5) from the normal cells (fractal dimension about 1.8). The Fourier transformed series showed fractal dimension results consistent with cell functions. A composite of breast cancer/normal cell matrix was built with cancer cell layers embedded within normal cell layers. The optical speckle pattern of a composite was investigated and computer modeling was used to extract the embedded cancer cell fractal dimension information. The measurement of the efficacy of a drug was simulated with the monitoring of the effect of added chemicals in the growth media. Laboratory optical speckle pattern monitoring of the effect of added chemicals was discussed. The extension for early cancer detection in mammography was also discussed and an example of the application of the anisotropic spatial variation of the fractal dimension via the Higuchi fractal method was presented.