The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathione S-transferase fusion protein of the trans-activation domain of activating transcription factor-2 (GST-ATF2-(1-115)) by p38 MAPK␣ (p38␣) as a model system for the phosphorylation of ATF2 by p38␣. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38␣ dissociates from GST-ATF2-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38␣ showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity, k cat /K M , of p38␣ for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38␣ in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate.

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The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A 2 (PLA 2 ), which catalyzes the release of sn -2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA 2 in mouse keratinocytes, a cytosolic or cPLA 2 and two secretory or sPLA 2 s in the membrane. The PLA 2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA 2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA 2 and at least one type of sPLA 2 . Inhibitor data further confirmed the identities of these PLA 2 s. The cPLA 2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4 ␣ -TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187and 4 ␣ -TPA-treated keratinocytes suggests activation of sPLA 2 . These activities may be due to the existence of both type I and type II sPLA 2 , as both were identified by polymerase chain reactions.In conclusion, keratinocytes express several forms of phospholipase A 2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.-Li-Stiles, B., H-H. Lo, and S. M. Fischer. Identification and characterization of several forms of phospholipase A 2 in mouse epidermal keratinocytes.

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.