The Acidic Region and Conserved Putative Protein Kinase C Phosphorylation Site in Nef Are Important for SIV Replication in Rhesus Macaques

We investigated effects of three kinds of putative protein kinase C (PKC) inhibitors, calphostin C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and stauro-sporine, on aortic muscle contractions induced by KCl, phenylephrine, 12-O-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13-dibutyrate (PDBu). Calphostin C noncompetitively inhibited TPA-induced contractions in a concentration-dependent manner. At 10(-6) M, calphostin C completely abolished responses to TPA and also effectively inhibited PDBu-induced contractions. Such a concentration of calphostin C had no effect on KCl-induced contractions but decreased the maximal tension of phenylephrine-induced response curve by 35.3 +/- 6.6% H-7 (10(-5) M had little effect on TPA-induced contraction but significantly inhibited contractile responses to phenylephrine and KCl. Staurosporine (10(-8) M, 3 x 10(-8) M) inhibited contractile responses to KCl, phenylephrine, and TPA. We suggest that staurosporine and H-7, which are known to act on the catalytic domain of PKC carrying high degree of sequence homology with other protein kinases, are relatively nonselective for PKC. On the other hand, calphostin C acting on the regulatory domain of PKC, which is distinct from other protein kinases, may serve as a relatively more selective PKC inhibitor.

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Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

Molecular Biology of the Cell ◽

10.1091/mbc.e08-11-1129 ◽

2009 ◽

Vol 20 (18) ◽

pp. 4120-4129 ◽

Cited By ~ 19

Author(s):

David Chamorro ◽

Lourdes Alarcón ◽

Arturo Ponce ◽

Rocio Tapia ◽

Héctor González-Aguilar ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Nuclear Export ◽

Phosphorylation Site ◽

Putative Protein ◽

Kinase C ◽

Junction Protein ◽

Zona Occludens ◽

Intracellular Fate ◽

Putative Protein Kinase

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCε. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

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