Morphine analgesic properties and side effects such as tolerance are mediated by the l opioid receptor (MOR) whose endocytosis is considered of primary importance for opioid pharmacological effects. Here, we show that p38 mitogen-activated protein kinase (MAPK) activation is required for MOR endocytosis and sufficient to trigger its constitutive internalization in the absence of agonist. Further studies established a functional link between p38 MAPK and the small GTPase Rab5, a key regulator of endocytosis. Expression of an activated mutant of Rab5 stimulated endocytosis of MOR ligand-independently in wild-type but not in p38aÀ/À cells. We found that p38a can phosphorylate the Rab5 effectors EEA1 and Rabenosyn-5 on Thr-1392 and Ser-215, respectively, and these phosphorylation events regulate the recruitment of EEA1 and Rabenosyn-5 to membranes. Moreover, phosphomimetic mutation of Thr-1392 in EEA1 can bypass the requirement for p38a in MOR endocytosis. Our results highlight a novel mechanism whereby p38 MAPK regulates receptor endocytosis under physiological conditions via phosphorylation of Rab5 effectors.

p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38␣, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38␣ correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38␣ ؊/؊ knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38␣-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38␣ ؊/؊ cells. Pharmacological inhibition of p38␣ also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38␣ into p38␣ ؊/؊ cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38␣؊/؊ cells and that the 3untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38␣-dependent manner. Our data indicate that p38␣ can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.

Interstrand cross-link (ICL) is a covalent modification of both strands of DNA, which prevents DNA strand separation during transcription and replication. Upon photoactivation 8-methoxypsoralen (8-MOP+UVA) alkylates both strands of DNA duplex at the 5,6-double bond of thymidines, generating monoadducts (MAs) and ICLs. It was thought that bulky DNA lesions such as MAs are eliminated only in the nucleotide excision repair pathway. Instead, non-bulky DNA lesions are substrates for DNA glycosylases and AP endonucleases which initiate the base excision repair (BER) pathway. Here we examined whether BER might be involved in the removal of psoralen–DNA photoadducts. The results show that in human cells DNA glycosylase NEIL1 excises the MAs in duplex DNA, subsequently the apurinic/apyrimidinic endonuclease 1, APE1, removes the 3′-phosphate residue at single-strand break generated by NEIL1. The apparent kinetic parameters suggest that NEIL1 excises MAs with high efficiency. Consistent with these results HeLa cells lacking APE1 and/or NEIL1 become hypersensitive to 8-MOP+UVA exposure. Furthermore, we demonstrate that bacterial homologues of NEIL1, the Fpg and Nei proteins, also excise MAs. New substrate specificity of the Fpg/Nei protein family provides an alternative repair pathway for ICLs and bulky DNA damage.