CaMK phosphatase (CaMKP/POPX2/PPM1F) inhibitors suppress the migration of human breast cancer MDA-MB-231 cells with loss of polarized morphology.

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.

CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation.

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.

Effect of heterocyclic pyrimidine compounds on UVB-induced cell damage in human keratinocytes and on melanogenesis in mouse B16 cells.

We investigated the protective effect of several heterocyclic pyrimidine compounds against ultraviolet B (UVB)-induced damage in human keratinocyte HaCaT cells, as well as the inhibitory effect on melanogenesis in B16 melanoma cells. One of the compounds examined, 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H)pyrrolo[3,4d]pyrimidine maleate (MS-818), showed low cytotoxicity even at 500 microM. At 50-500 microM, MS-818 dose-dependently suppressed the UVB (100 mJ/cm(2))-induced elevation of tumor necrosis factor alpha (TNF-alpha), one of the trigger cytokines for cell death, in HaCaT cells. In addition, MS-818 (100 microM) markedly inhibited melanogenesis in B16 melanoma cells via downregulation of tyrosinase expression mediated by microphthalmia-associated transcription factor (MITF) and extracellular signal-regulated kinase (ERK). In conclusion, MS-818 protected epidermal cells from UVB-induced damage and also suppressed melanogenesis in melanoma cells. It appears to be a good candidate for a new UVB-protective and whitening agent for application in cosmetics.