Adaptative Value of a PKC?PKI55 Feedback Loop of Inhibition That Prevents the Kinase?s Deregulation
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Article: Adaptative Value of a PKC?PKI55 Feedback Loop of Inhibition That Prevents the Kinase?s Deregulation
- SourceAvailable from: Rita Selvatici
[Show abstract] [Hide abstract] ABSTRACT: We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1-16 (peptide 5), 1-8 (peptide 8) and 1-5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse. The peptides 5, 8 and 9 (0.1 and 10 nmol i.c.v.) were effective in both the parameters chosen to test the anti-inflammatory activity, i.e., the xylene-induced ear edema and the acetic acid-induced infiltration of neutrophils in the peritoneal cavity. In addition, they displayed analgesic effect, evaluated by the acetic acid-induced writhing test. All the peptides' effects were shared by the reference compounds, dexamethasone and indomethacin (10 mg kg(-1) i.p.), but not by the 9-scramble peptide (10 nmol i.c.v.). The peptide 9, which represents the shortest active sequence of the PKI55 protein, was tested in the ear edema model even following intraperitoneal (i.p.) administration and proved to be effective in the range doses 3-30 mg kg(-1). Moreover, an increase in plasma corticosterone levels was detected in mice treated with the peptide 9, but not with the 9-scramble peptide (both at 10 nmol i.c.v.). The anti-inflammatory and analgesic effects of the PKI55-derived synthetic peptides, possibly related both to PKC inhibition and hypothalamic-pituitary-adrenal axis activation, deserve further investigation in view of potential therapeutic exploitation.
- "In recent years, many academics, medicinal chemists and pharmaceutical researchers have been involved in the search for new molecules able to interfere in neutrophil upregulation, in order to exploit their therapeutic potentials (Cesarini et al. 2009; Mackay 2008). In this context, the present study investigated the potential anti-inflammatory role of the synthetic peptides 5, 8 and 9, derived from PKI55, an endogenous PKC inhibitor protein (Selvatici et al. 2003, 2007, 2008; Borgatti et al. 2009), using experimental approaches in vivo. The results show that, in the mouse, the i.c.v. "
[Show abstract] [Hide abstract] ABSTRACT: The PKI55 protein was identified in our laboratory as specific protein kinase C inhibitor. We previously demonstrated that PKI55 is poorly translated in vivo and acts promoting PKC degradation and establishing a feedback loop of inhibition. However, our understanding of mechanisms by which the expression of PKI55 is regulated, is limited. In the present work we investigated the mRNA expression of PKI55 in human tissues by Northern blotting and RT-PCR, demonstrating that it is highly expressed in brain tissue. Moreover, since the computational analysis of the gene promoter region showed two sites (Box 1 and Box 2) similar to consensus sequences for AP1 and GAGA factors, we investigated their ability to bind to these proteins. Electrophoretic mobility shift assays showed that GAGA factors preferentially interacted with Box 2, while AP1 elements linked preferentially Box 1 sequence. We suggest that the interaction of these transcription factors with Box 1 and Box 2 could regulate the transcription of the PKI55 gene and, consequently, the expression of PKC.
- "When 12 mg of crude nuclear extracts from human leukemic K562 cells were incubated for 20 min in the presence of the cold double-stranded Box1 or AP1 (representative AP1 consensus sequence), a concentration-dependent inhibition of interactions between 32 P-labeled Box 1 sequence and nuclear factors was observed (Fig. 6A, left side of the panel). On the contrary, when 32 P-labeled Box 2 mers were used, cold Box 2, but not AP1 oligonucleotides, exhibited inhibitory activity with respect Fig. 2. PKI55 coding sequence and regulatory region of BAC Clone BACRPCI11-1064L18 from 87 205 to 88 803 nucleotides . The PKI55 coding sequence spans between 88 535 and 88 701 bp and possesses both starting and terminal codons. "
- [Show abstract] [Hide abstract] ABSTRACT: PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.