Lkb1/Stk11 regulation of mTOR signaling controls the transition of chondrocyte fates and suppresses skeletal tumor formation.

Liver kinase b1 (Lkb1) protein kinase activity regulates cell growth and cell polarity. Here, we show Lkb1 is essential for maintaining a balance between mitotic and postmitotic cell fates in development of the mammalian skeleton. In this process, Lkb1 activity controls the progression of mitotic chondrocytes to a mature, postmitotic hypertrophic fate. Loss of this Lkb1-dependent switch leads to a dramatic expansion of immature chondrocytes and formation of enchondroma-like tumors. Pathway analysis points to a mammalian target of rapamycin complex 1-dependent mechanism that can be partially suppressed by rapamycin treatment. These findings highlight a critical requirement for integration of mammalian target of rapamycin activity into developmental decision-making during mammalian skeletogenesis.

SAD-A kinase controls islet ß-cell size and function as a mediator of mTORC1 signaling.

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet ß-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic ß-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet ß-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines ß-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet ß-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

SAD-A potentiates glucose-stimulated insulin secretion as a mediator of glucagon-like peptide 1 response in pancreatic ß cells.

Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic ß cells, which can be restored by glucagon-like peptide 1 (GLP-1), an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet ß cells remain poorly understood. Here we investigated a role of SAD-A, an AMP-activated protein kinase (AMPK)-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretin's effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS and further potentiated GLP-1's effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is expressed exclusively in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through cyclic AMP (cAMP)/Ca(2+)-dependent signaling pathways in islet ß cells. Furthermore, we identified Thr443 as a key autoinhibitory phosphorylation site which mediates SAD-A's effect on incretin response in islet ß cells. Consequently, ablation of Thr443 significantly enhanced GLP-1's effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet ß cells.

Derlin-2-deficient mice reveal an essential role for protein dislocation in chondrocytes.

Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the endoplasmic reticulum (ER). Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated conditional knockout mice to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2-deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole-body deletion of Derlin-2 results in perinatal death. The few mice that survived to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes.

LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons.

The polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. We show here that the serine/threonine kinase LKB1, previously implicated in the establishment of epithelial polarity and control of cell growth, is required for axon specification during neuronal polarization in the mammalian cerebral cortex. LKB1 polarizing activity requires its association with the pseudokinase Stradalpha and phosphorylation by kinases such as PKA and p90RSK, which transduce neurite outgrowth-promoting cues. Once activated, LKB1 phosphorylates and thereby activates SAD-A and SAD-B kinases, which are also required for neuronal polarization in the cerebral cortex. SAD kinases, in turn, phosphorylate effectors such as microtubule-associated proteins that implement polarization. Thus, we provide evidence in vivo and in vitro for a multikinase pathway that links extracellular signals to the intracellular machinery required for axon specification.

SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER.

Protein quality control in the endoplasmic reticulum (ER) involves recognition of misfolded proteins and dislocation from the ER lumen into the cytosol, followed by proteasomal degradation. Viruses have co-opted this pathway to destroy proteins that are crucial for host defense. Examination of dislocation of class I major histocompatibility complex (MHC) heavy chains (HCs) catalyzed by the human cytomegalovirus (HCMV) immunoevasin US11 uncovered a conserved complex of the mammalian dislocation machinery. We analyze the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process. Perturbation of SEL1L function discriminates between the dislocation pathways used by US11 and US2, which is a second HCMV protein that catalyzes dislocation of class I MHC HCs. Furthermore, reduction of the level of SEL1L by small hairpin RNA (shRNA) inhibits the degradation of a misfolded ribophorin fragment (RI332) independently of the presence of viral accessories. These results allow us to place SEL1L in the broader context of glycoprotein degradation, and imply the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER.

Murine polyomavirus requires the endoplasmic reticulum protein Derlin-2 to initiate infection.

The pathways by which viruses enter cells are diverse, but in all cases, infection necessitates the transfer of the viral genome across a cellular membrane. Polyomavirus (Py) particles, after binding to glycolipid and glycoprotein receptors at the cell surface, are delivered to the lumen of the endoplasmic reticulum (ER). The nature and extent of virus disassembly in the ER, how the viral genome is transported to the cytosol and subsequently to the nucleus, and whether any cellular proteins are involved are not known. Here, we identify an ER-resident protein, Derlin-2, a factor implicated in the removal of misfolded proteins from the ER for cytosolic degradation, as a component of the machinery required for mouse Py to establish an infection. Inhibition of Derlin-2 function by expression of either a dominant-negative form of Derlin-2 or a short hairpin RNA that reduces Derlin-2 levels blocks Py infection by 50 to 75%. The block imposed by Derlin-2 inhibition occurs after the virus reaches the ER and can be bypassed by the introduction of Py DNA into the cytosol. These findings suggest a mode of Py entry that involves cytosolic access via the quality control machinery in the ER.

Signal peptide peptidase is required for dislocation from the endoplasmic reticulum.

Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.

Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane.

Polypeptides that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated from the ER membrane to the cytosol where they are degraded by the proteasome. Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, was identified as a factor that is required for the human cytomegalovirus US11-mediated dislocation of class I MHC heavy chains from the ER membrane to the cytosol. Derlin-1 acts in concert with the AAA ATPase p97 to remove dislocation substrate proteins from the ER membrane, but it is unknown whether other factors aid Derlin-1 in its function. Mammalian genomes encode two additional, related proteins (Derlin-2 and Derlin-3). The similarity of the mammalian Derlin-2 and Derlin-3 proteins to yeast Der1p suggested that these as-yet-uncharacterized Derlins also may play a role in ER protein degradation. We demonstrate here that Derlin-2 is an ER-resident protein that, similar to Derlin-1, participates in the degradation of proteins from the ER. Furthermore, we show that Derlin-2 forms a robust multiprotein complex with the p97 AAA ATPase as well as the mammalian orthologs of the yeast Hrd1p/Hrd3p ubiquitin-ligase complex. The data presented here define a set of interactions between proteins involved in dislocation of misfolded polypeptides from the ER.

Human cytomegalovirus protein US11 provokes an unfolded protein response that may facilitate the degradation of class I major histocompatibility complex products.

The human cytomegalovirus (HCMV) glycoprotein US11 diverts class I major histocompatibility complex (MHC) heavy chains (HC) from the endoplasmic reticulum (ER) to the cytosol, where HC are subjected to proteasome-mediated degradation. In mouse embryonic fibroblasts that are deficient for X-box binding protein 1 (XBP-1), a key transcription factor in the unfolded protein response (UPR) pathway, we show that degradation of endogenous mouse HC is impaired. Moreover, the rate of US11-mediated degradation of ectopically expressed HLA-A2 is reduced when XBP-1 is absent. In the human astrocytoma cell line U373, turning on expression of US11, but not US2, is sufficient to induce a UPR, as manifested by upregulation of the ER chaperone Bip and by splicing of XBP-1 mRNA. In the presence of dominant-negative versions of XBP-1 and activating transcription factor 6, the kinetics of class I MHC HC degradation were delayed when expression of US11 was turned on. The magnitude of these effects, while reproducible, was modest. Conversely, in cells that stably express high levels of US11, the degradation of HC is not affected by the presence of the dominant negative effectors of the UPR. An infection of human foreskin fibroblasts with human cytomegalovirus induced XBP-1 splicing in a manner that coincides with US11 expression. We conclude that the contribution of the UPR is more pronounced on HC degradation shortly after induction of US11 expression and that US11 is sufficient to induce such a response.

A membrane protein required for dislocation of misfolded proteins from the ER.

After insertion into the endoplasmic reticulum (ER), proteins that fail to fold there are destroyed. Through a process termed dislocation such misfolded proteins arrive in the cytosol, where ubiquitination, deglycosylation and finally proteasomal proteolysis dispense with the unwanted polypeptides. The machinery involved in the extraction of misfolded proteins from the ER is poorly defined. The human cytomegalovirus-encoded glycoproteins US2 and US11 catalyse the dislocation of class I major histocompatibility complex (MHC) products, resulting in their rapid degradation. Here we show that US11 uses its transmembrane domain to recruit class I MHC products to a human homologue of yeast Der1p, a protein essential for the degradation of a subset of misfolded ER proteins. We show that this protein, Derlin-1, is essential for the degradation of class I MHC molecules catalysed by US11, but not by US2. We conclude that Derlin-1 is an important factor for the extraction of certain aberrantly folded proteins from the mammalian ER.

Dislocation of a type I membrane protein requires interactions between membrane-spanning segments within the lipid bilayer.

The human cytomegalovirus gene product US11 causes rapid degradation of class I major histocompatibility complex (MHCI) heavy chains by inducing their dislocation from the endoplasmic reticulum (ER) and subsequent degradation by the proteasome. This set of reactions resembles the endogenous cellular quality control pathway that removes misfolded or unassembled proteins from the ER. We show that the transmembrane domain (TMD) of US11 is essential for MHCI heavy chain dislocation, but dispensable for MHCI binding. A Gln residue at position 192 in the US11 TMD is crucial for the ubiquitination and degradation of MHCI heavy chains. Cells that express US11 TMD mutants allow formation of MHCI-beta2m complexes, but their rate of egress from the ER is significantly impaired. Further mutagenesis data are consistent with the presence of an alpha-helical structure in the US11 TMD essential for MHCI heavy chain dislocation. The failure of US11 TMD mutants to catalyze dislocation is a unique instance in which a polar residue in the TMD of a type I membrane protein is required for that protein's function. Targeting of MHCI heavy chains for dislocation by US11 thus requires the formation of interhelical hydrogen bonds within the ER membrane.