STRAD Pseudokinases Regulate Axogenesis and LKB1 Stability

BACKGROUND: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and β. The roles of STRADα and STRADβ in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions. RESULTS: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization. CONCLUSIONS: We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability

Kinases and pseudokinases: Lessons from RAF

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be “frozen” by introducing mutations at their hydrophobic spines

AMP as a Low-Energy Charge Signal Autonomously Initiates Assembly of AXIN-AMPK-LKB1 Complex for AMPK Activation

The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis by sensing cellular energy status. AMPK is mainly activated via phosphorylation by LKB1 when cellular AMP/ADP levels are increased. However, how AMP/ADP brings about AMPK phosphorylation remains unclear. Here, we show that it is AMP, but not ADP, that drives AXIN to directly tether LKB1 to phosphorylate AMPK. The complex formation of AXIN-AMPK-LKB1 is greatly enhanced in glucose-starved or AICAR-treated cells and in cell-free systems supplemented with exogenous AMP. Depletion of AXIN abrogated starvation-induced AMPK-LKB1 colocalization. Importantly, adenovirus-based knockdown of AXIN in the mouse liver impaired AMPK activation and caused exacerbated fatty liver after starvation, underscoring an essential role of AXIN in AMPK activation. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.http://news.xmu.edu.cn/s/13/t/542/22/a9/info139945.ht

A secretory kinase complex regulates extracellular protein phosphorylation.

Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation

Characterisation of an alternative splice variant of LKB1

The LKB1 protein kinase has previously been implicated in a number of major physiological processes including cell proliferation, polarity and energy metabolism. LKB1 is an upstream kinase for AMP-activated protein kinase (AMPK) and 12 members of the AMPK-related family of kinases. In this study, western blotting of LKB1 from various mouse tissues indicated the existence of two molecular weight bands in testis. This, along with the presence of a potential alternative exon at the 3’ end of the gene, led to the discovery that an alternatively-spliced variant of LKB1 is present in mice. This new splice variant has been termed the ‘short form’ (LKB1S) and the original protein termed the ‘long form’ (LKB1L). The proteins are identical in sequence apart from the C-terminus. The overall aim of this study was to characterise the LKB1S protein; its activity, regulation and possible physiological function, using overexpression studies in mammalian cells and analysis of mice lacking LKB1S. An LKB1S-specific antibody was used to show that LKB1S is primarily expressed in testis. It was demonstrated, using overexpression studies, that both forms can activate AMPK, form an active complex with the regulatory proteins STRADα, STRADβ and MO25 and have a similar sub-cellular localisation. The C-terminus of mouse/human LKB1L can be phosphorylated at serine 431/428 (Sapkota et al., 2001). This residue is absent from LKB1S and so the effect of phosphorylation was investigated to determine if this could lead to differences in activity between the splice forms. However, the results suggest that phosphorylation at this site does not affect the ability of LKB1L to activate AMPK in cells. Male mice lacking expression of LKB1S are infertile. The mice display severe sperm abnormalities and sperm counts are significantly reduced. Histological analysis of testis showed that although early spermatogenesis appears to progress normally, abnormalities become apparent later on, culminating in very few spermatozoa reaching the epididymis. One possible reason that has been investigated is that there is a defect in the release of mature sperm from the seminiferous epithelium (spermiation)

Novel Posttranslational Modification in LKB1 Activation and Function

Cancer cells display dramatic alterations in cellular metabolism to meet their needs of increased growth and proliferation. In the last decade, cancer research has brought these pathways into focus, and one emerging issue that has come to attention is that many oncogenes and tumor-suppressors are intimately linked to metabolic regulation (Jones and Thompson, 2009). One of the key tumor-suppressors involved in metabolism is Liver Kinase B1 (LKB1). LKB1 is the major upstream kinase of the evolutionarily conserved metabolic sensor—AMP-activated protein kinase (AMPK). Activation of the LKB1/AMPK pathway provides a survival advantage for cells under energy stress. LKB1 forms a heterotrimeric complex and is activated through binding of the two regulatory proteins, STRAD and MO25. LKB1 has been shown to be a tumor-suppressor in various mouse models; however, recent studies suggest that LKB1 has pro-oncogenic functions. How the LKB1 activity and the LKB1-STRAD-MO25 complex are maintained and regulated and how LKB1 regulates cancer development are largely unclear. Here we show that K63-linked LKB1 polyubiquitination by the Skp1-Cul1-F-box-protein/Skp2 (Skp2-SCF) ubiquitin ligase complex is critical for LKB1 activation by a mechanism of maintaining the LKB1-STRAD-MO25 complex integrity. We further demonstrate that oncogenic Ras acts upstream of Skp2 to promote LKB1 polyubiquitination by activating the Skp2-SCF ubiquitin ligase complex. Moreover, Skp2-mediated LKB1 polyubiquitination is required for energy stress-induced cell survival. We also detected upregulation and positive correlation of Skp2 and LKB1 expression in late-stage hepatocellular carcinoma (HCC), and their overexpression predicts poor survival outcome of HCC patients. Finally, we show that Skp2-mediated LKB1 polyubiquitination is important for HCC tumor growth in a mouse subcutaneous xenograft tumor model. Our study provides new insights into the upstream regulation of LKB1 activation and suggests a potential target, the Ras/Skp2/LKB1 axis, for cancer therapy

AMP-Activated Protein Kinase:Do We Need Activators or Inhibitors to Treat or Prevent Cancer?

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance. In response to metabolic stress, it acts to redress energy imbalance through promotion of ATP-generating catabolic processes and inhibition of ATP-consuming processes, including cell growth and proliferation. While findings that AMPK was a downstream effector of the tumour suppressor LKB1 indicated that it might act to repress tumourigenesis, more recent evidence suggests that AMPK can either suppress or promote cancer, depending on the context. Prior to tumourigenesis AMPK may indeed restrain aberrant growth, but once a cancer has arisen, AMPK may instead support survival of the cancer cells by adjusting their rate of growth to match their energy supply, as well as promoting genome stability. The two isoforms of the AMPK catalytic subunit may have distinct functions in human cancers, with the AMPK-α1 gene often being amplified, while the AMPK-α2 gene is more often mutated. The prevalence of metabolic disorders, such as obesity and Type 2 diabetes, has led to the development of a wide range of AMPK-activating drugs. While these might be useful as preventative therapeutics in individuals predisposed to cancer, it seems more likely that AMPK inhibitors, whose development has lagged behind that of activators, would be efficacious for the treatment of pre-existing cancers

Investigation of LKB1 Ser⁴³¹ phosphorylation and Cys⁴³³ farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity

The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser(431)) by PKA (cAMP-dependent protein kinase) and RSK (ribosomal S6 kinase) and farnesylated (Cys(433)) within a CAAX motif. To better define the role that these post-translational modifications play, we created homozygous LKB1(S431A/S431A) and LKB1(C433S/C433S) knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1(C433S/C433S) mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1(S431A/S431A) animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1(C433S/C433S) mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1(C433S/C433S) mice. The activity of several of the AMPK-related kinases analysed [BRSK1 (BR serine/threonine kinase 1), BRSK2, NUAK1 (NUAK family, SNF1-like kinase 1), SIK3 (salt-inducible kinase 3) and MARK4 (MAP/microtubule affinity-regulating kinase 4)] was not affected in tissues derived from LKB1(S431A/S431A) or LKB1(C433S/C433S) mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the plasma membrane as a result of myristoylation of its regulatory AMPKβ subunit. This raises the possibility that LKB1 farnesylation and myristoylation of AMPKβ might promote the interaction and co-localization of these enzymes on a two-dimensional membrane surface and thereby promote efficient activation of AMPK

Insights From Pretzel Syndrome: The Role of STRADA in Neuronal Migration and Cortical Development

Pretzel Syndrome (also Polyhydramnios, Megalencephaly, and Symptomatic Epilepsy syndrome; PMSE) is a recently described rare neurodevelopmental disorder occurring in the Old Order Mennonite pediatric population, and characterized by intractable infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter, suggestive of failed neuronal migration. PMSE is caused by a homozygous deletion of exons 9-13 of LYK5/STRADA, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin (mTOR). Therefore, we hypothesize that STRADA plays a critical role in neuronal migration through modulating mTOR (specifically mTOR complex 1, mTORC1) signaling, and that therapeutic mTORC1 inhibition can ameliorate features of the PMSE disease phenotype. To test this hypothesis, we model PMSE in vitro using stable shRNA knockdown of STRADA (STRADA KD) in mouse neural progenitor cells (mNPCs). In vivo, we use in utero electroporation to create focal STRADA KD in the developing mouse brain. We show that STRADA depletion disrupts pathfinding and polarization in migrating mNPCs in vitro, and this effect can be rescued by inhibition of mTORC1 with rapamycin or of its downstream effector p70S6kinase (p70S6K) with PF-4708671 (p70S6Ki), indicating an mTORC1-specific dependence. We then define a pathway for this effect downstream of mTORC1, through insulin receptor substrate 1 (IRS1) signaling to cofilin, and finally modulating actin dynamics. In vivo, we demonstrate that STRADA KD causes a cortical lamination defect in the mouse, which can be rescued with rapamycin treatment, confirming the dependence of STRADA\u27s effect on mTORC1 signaling and suggesting an important target for patient therapy. To correlate our mouse model with PMSE, we demonstrate congruent mTORC1 and downstream signaling and rescue of migration deficit with rapamycin and p70S6Ki in PMSE patient fibroblasts. Finally, we report reduction of seizure frequency with rapamycin treatment in previously intractable PMSE patients. Our findings define a novel role for STRADA in neuronal migration, demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition can serve as an effective therapeutic bio-target in PMSE as well as other devastating mTOR-associated neurodevelopmental disorders