Fatima Lekmine

Activation of Protein Kinase Cδ by IFN-γ

Engagement of the type II IFN (IFN-γ) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-γ-activated site (GAS) elements in the promoters of IFN-γ-inducible genes. An important event in IFN-γ-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3′-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3′-kinase and is engaged in IFN-γ signaling. Our data demonstrate that PKCδ is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKCδ associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKCδ activity diminishes phosphorylation of Stat1 on Ser727 and IFN-γ-dependent transcriptional regulation via IFN-γ-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKCδ is activated during engagement of the IFN-γ receptor and plays an important role in IFN-γ signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-γ-stimulated genes.

Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G2-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents. [Mol Cancer Ther 2009;8(10):2947–58]

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt

Amino acids 50–77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin’s preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

Engagement of the CrkL adaptor in interferon α signalling in BCR-ABL-expressing cells

Interferon α (IFNα) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR–ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR–ABL‐expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNα‐signalling in the CML‐derived KT‐1 cell line, which expresses BCR–ABL and is sensitive to the growth inhibitory effects of IFNα. CrkL is constitutively associated with BCR–ABL in these cells and treatment with IFNα had no effect on the BCR–ABL/CrkL interaction. After IFNα stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNα‐dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N‐terminus SH3 domains of CrkL. The resulting CrkL–Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the β‐casein promoter or the promoter of the PML gene, an IFNα‐inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT‐1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR–ABL in CML‐derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti‐proliferative effects of IFNα in CML.