The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

Individuals with type 2 diabetes have an increased risk of atherosclerosis. One factor underlying this is dyslipidemia, which in hyperinsulinemic subjects with early type 2 diabetes is typically characterized by increased VLDL secretion but normal LDL cholesterol levels, possibly reflecting enhanced catabolism of LDL via hepatic LDLRs. Recent studies have also suggested that hepatic insulin signaling sustains LDLR levels. We therefore sought to elucidate the mechanisms linking hepatic insulin signaling to regulation of LDLR levels. In WT mice, insulin receptor knockdown by shRNA resulted in decreased hepatic mTORC1 signaling and LDLR protein levels. It also led to increased expression of PCSK9, a known post-transcriptional regulator of LDLR expression. Administration of the mTORC1 inhibitor rapamycin caused increased expression of PCSK9, decreased levels of hepatic LDLR protein, and increased levels of VLDL/LDL cholesterol in WT but not Pcsk9 -/-mice. Conversely, mice with increased hepatic mTORC1 activity exhibited decreased expression of PCSK9 and increased levels of hepatic LDLR protein levels. Pcsk9 is regulated by the transcription factor HNF1α, and our further detailed analyses suggest that increased mTORC1 activity leads to activation of PKCδ, reduced activity of HNF4α and HNF1α, decreased PCSK9 expression, and ultimately increased hepatic LDLR protein levels, which result in decreased circulating LDL levels. We therefore suggest that PCSK9 inhibition could be an effective way to reduce the adverse side effect of increased LDL levels that is observed in transplant patients taking rapamycin as immunosuppressive therapy.

Background Autosomal dominant hypercholesterolemia (ADH), characterized by elevated plasma levels of low density lipoprotein-cholesterol (LDL-C), is caused by variants in at least three different genes:LDL receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin-like kexin type 9 (PCSK9). There is paucity of data about the molecular basis of ADH among ethnic groups other than those of European or Japanese descent. Here, we examined the molecular basis of ADH in a multi-ethnic patient cohort from lipid clinics in a large urban U.S. city. Methods and Results A total of 38 males and 53 females, age 22 to 76 years, met modified Simon-Broome criteria for ADH and were screened for mutations in the exons and consensus splice sites of LDLR, and in selected exons of APOB and PCSK9. Deletions and duplications of LDLR exons were detected with multiplex ligation-dependent probe amplification. Heterozygous variants in LDLR were identified in 30 patients and in APOB in one patient. The remaining 60 patients (65%) had “unexplained ADH.” A higher proportion of African Americans (77%) than either non-Hispanic whites (57%) or Hispanics (53%) had “unexplained ADH.” As compared to patients with LDLR variants, those with “unexplained ADH” had lower levels of LDL-C (292 ± 47vs 239 ± 42 mg/dL, respectively; p < 0.0001) and higher levels of HDL-cholesterol (45 ± 12vs 54 ± 13 mg/dL, respectively, p = 0.003). Conclusions Our findings suggest that additional loci may contribute to ADH, especially in understudied populations such as African Americans.

The Raf-MEK 2 -ERK kinase cascade is one of the most extensively studied signal transduction modules on account of its core participation in both normal and pathological cell regulatory networks. A great deal of information has been gathered about the acute participation of the Raf-MEK-ERK signal transduction circuitry in the modulation and/or propagation of a broad range of dynamic cell regulatory events ranging from cellular proliferation and tumorigenesis to differentiation and cell specialization (1-3). Attention is now necessarily becoming focused on understanding the molecular architecture that allows the core enzymatic components of this cascade to be shared among multiple regulatory pathways yet produce distinct biological outcomes in response to discrete inductive signals. There is accumulating evidence that control of the amplitude, duration, and subcellular compartmentalization of ERK activation are all critical determinants of the biological response (4 -7). The discovery and characterization of non-catalytic accessory components, or scaffolds, that generate higher order molecular organization to modulate the assembly, activation, and compartmentalization of MAPK cascades are beginning to reveal mechanisms that generate specificity in the coupling of MAPK activation to appropriate biological responses (8 -11).We have recently described IMP (impedes mitogenic signal propagation) as a specifier of ERK activation amplitude through inhibition of functional coupling between Raf and MEK kinases (12). The mode of action of IMP is dependent upon the presence of the Raf-MEK-ERK scaffolding protein KSR1 (kinase suppressor of Ras 1) and is a consequence of impairing the capacity of this scaffold to promote signal propagation through Raf to MEK (13). Here, we have examined how IMP association with KSR1 can limit Raf⅐MEK⅐ERK complex formation and function. We find that KSR1 homo-oligomerization is required to couple distinct KSR⅐MEK and KSR⅐B-Raf complexes to allow MEK activation. Furthermore, KSR1 promotes formation of B-Raf/c-Raf hetero-oligomers that contribute to c-Raf kinase activation. IMP association blocks both KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization to impair both MEK activation and the contribution of Raf oligomers to c-Raf kinase activation. These observations reveal the participation of KSR1 both upstream and downstream of Raf kinase activation in human cells and highlight Raf family oligomerization as a basic control point for modulation of signal amplitude by IMP. EXPERIMENTAL PROCEDURESCell Culture and Transfection-HEK293 cells were cultured in Dulbecco's modified Eagle's medium without sodium pyruvate (Invitrogen) with 10% fetal bovine serum and transfected with Lipofectamine and Plus Reagent (Invitrogen). HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and RNA interference was performed with DharmaFECT 1 (Dharmacon).siRNAs, Plasmids, and Antibodies-The following siRNA sequences were used: IMP-FW (GGACACAGCAGAG-GAAAU...