Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.

https://www.physiology.org/doi/pdf/10.1152/ajplung.2000.279.3.L429

Protein kinase C isozymes and the regulation of diverse cell responses

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal transduction pathways and the BCR-ABL oncoprotein in leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. These pathways have evolved regulatory proteins, which serve to limit their proliferative and antiapoptotic effects. Small molecular weight cell membrane-permeable drugs that target these pathways have been developed for leukemia therapy. One such example is imatinib mesylate, which targets the BCR-ABL kinase as well as a few structurally related kinases. This drug has proven to be effective in the treatment of CML patients. However, leukemic cells have evolved mechanisms to become resistant to this drug. A means to combat drug resistance is to target other prominent signaling components involved in the pathway or to inhibit BCR-ABL by other mechanisms. Treatment of imatinib-resistant leukemia cells with drugs that target Ras (farnysyl transferase inhibitors) or with the protein destabilizer geldanamycin has proven to be a means to inhibit the growth of resistant cells. This review will tie together three important signal transduction pathways involved in the regulation of hematopoietic cell growth and indicate how their expression is dysregulated by the BCR-ABL oncoprotein.

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis.

The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C δ) were Src family substrates whereas others (for example, phospholipase C-γ) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor Signaling

The Ets1 proto-oncoprotein is a member of the Ets family of transcription factors that share a unique DNA binding domain, the Ets domain. The DNA binding activity of Ets1 is controlled by kinases and transcription factors. Some transcription factors, such as AML-1, regulate Ets1 by targeting its autoinhibitory module. Others, such as Pax-5, alter Ets1 DNA binding properties. Ets1 harbors two phosphorylation sites, threonine-38 and an array of serines within the exon VII domain. Phosphorylation of threonine-38 by ERK1/2 activates Ets1, whereas phosphorylation of the exon VII domain by CaMKII or MLCK inhibits Ets1 DNA binding activity. Ets1 is expressed by numerous cell types. In haemotopoietic cells, it contributes to the regulation of cellular differentiation. In a variety of other cells, including endothelial cells, vascular smooth muscle cells and epithelial cancer cells, Ets1 promotes invasive behavior. Regulation of MMP1, MMP3, MMP9 and uPA as well as of VEGF and VEGF receptor gene expression has been ascribed to Ets1. In tumors, Ets1 expression is indicative of poorer prognosis.

/pdf/the-biology-of-the-ets1-proto-oncogene-1tu9pwg569.pdf

The Biology of the Ets1 Proto-Oncogene

The role of PKC isoforms in signal transduction pathways involved in regulation of the cell cycle, apoptosis, angiogenesis, differentiation, invasiveness, senescence and drug efflux are reviewed, along with the clinical results on the current crop of PKC inhibitors, including midostaurin (PKC-412, CGP 41251, N -benzoylstaurosporine), UCN-01 (7-hydroxystaurosporine), bryostatin 1, perifosine, ilmofosine, Ro 31-8220, Ro 32-0432, GO 6976, ISIS-3521 (CGP 64128A) and the macrocyclic bis (indolyl) maleimides (LY-333531, LY-379196, LY-317615). An appreciation of the complex, often contradictory roles of PKC isoforms in signal transduction pathways involved in cancer is important for interpreting the clinical results observed with PKC inhibitors of varying selectivity. An antisense oligonucleotide, ISIS-3521 and two orally available small molecule inhibitors, LY 333531 and midostaurin, have now advanced to latter stage development for cancer and/or other indications. These compounds have varying levels of selectivity for the PKC isoforms and for the kinase and initial safety and early clinical efficacy have been encouraging. At this stage, the potential of PKC inhibition for the treatment of cancer has not been fully realised. The concurrent inhibition of multiple PKC isoforms may yet provide an improved clinical outcome in treating cancers in view of the complex interrelated roles of the PKC isoforms.

Protein kinase C inhibitors as novel anticancer drugs

Protein kinase C (PKC) is a family of enzymes that play a ubiquitous role in intracellular signal transduction. Our understanding of the precise role of PKC has evolved considerably as a result of improved methodology and a better understanding of the signal transduction pathways. A number of primary pathways previously attributed to PKC have been re-examined and found to involve other kinases as our understanding of the PKC isozymes has evolved. PKC isozymes appear to play distinct, and in some cases opposing roles in the transduction of intracellular signals. The development of potent and selective PKC inhibitors, including isozyme-selective inhibitors, has opened new avenues for biochemical and pharmaceutical studies. The role of PKC in some of the pathways relevant to cardiovascular, peripheral microvascular, CNS, oncology, immune and infectious disease states are surveyed. A survey of the current generation of potent and selective ATP-competitive inhibitors is provided. The progress of PKC inhibitors currently in clinical development, including LY333531, ISIS 3521 (CGP 64128A), bryostatin 1, GF109203x, Ro 32-0432 and Ro 31-8220, Go 6976 and Go 7611, CPR 1006, and balanol (SPC 100840) are discussed.

Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development.

Protein Kinase C in the Treatment of Disease: Signal Transduction Pathways, Inhibitors, and Agents in Development

A method for inhibiting VEGF stimulated endothelial cell growth, such as associated with neoplasia, and VEGF stimulated capillary permeability, such as associated with pulmonary edema are disclosed, particularly using the β-isozyme selective PKC inhibitor, (S)-3,4-[N,N'-1,1'-((2''-ethoxy)-3'''(O)-4'''-(N,N-dimethylamino)-butane)-bis-(3,3'-indolyl)]-1(H)-pyrrole-2,5-dione hydrochloride salt.

Therapeutic treatment for VEGF related diseases

A method for treating neoplasms is disclosed, particularly using the β-isozyme selective PKC inhibitor, (S)-3,4-[N,N'-1,1'-((2''-ethoxy)-3'''(O)-4'''-(N,N-dimethylamino)-butane)-bis-(3,3'-indolyl)]-1(H)-pyrrole-2,5-dione or one of its salts, such PKC inhibitors enhance the clinical efficacy of oncolytic agents and radiation therapy.

Jirousek Michael R

Papers

Protein kinase C inhibitors as novel anticancer drugs

Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development.

Protein Kinase C in the Treatment of Disease: Signal Transduction Pathways, Inhibitors, and Agents in Development

Therapeutic treatment for VEGF related diseases

Use of protein kinase c inhibitors to enhance the clinical efficacy of oncolytic agents and radiation therapy