Cyclin D1 plays a critical role in controlling the G 1 /S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2␣ (eIF2␣) kinases. We examined the effect of activation of the eIF2␣ kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2␣ phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3␤ and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2␣ phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2␣ phosphorylation during short, but not prolonged, periods of stress.

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2␣ at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2␣. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2␣ in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress.

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A20 IIA1.6 B cells cotransfected with FcαR and wild-type γ-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or FcαR and γ-chain, in which the wt-ITAM was substituted with the FcγRIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of FcαR-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented FcαR-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that FcαR may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B α (PKBα) activation. Cross-linking FcαR on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKBα. Furthermore, FcαR cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKBα to capped cell surface FcαR irrespective of the γ-chain ITAM. Although FcαR endocytosis was accompanied by translocation of PDK1 and phospho-PKBα to FcαR-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKBα remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKBα translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.