Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKdelta containing a sterile alpha-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKdelta formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKdelta. Furthermore, we found that DGKdelta was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKdelta mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKdelta. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKdelta and that DGKdelta is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.     

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.     

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.     

Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.     

Diacylglycerol kinases: emerging downstream regulators in cell signaling systems

Diacylglycerol kinase (DGK) regulates signal transduction by modulating the balance between the two signaling lipids, diacylglycerol and phosphatidic acid. DGK and its homologs occur in a wide range of multicellular organisms and the mammalian DGK is known to consist of nine members with a considerable incidence of alternative splicing. Recent work has established that DGK serves as a key attenuator of diacylglycerol of signaling functions and that the mammalian isozymes are equipped with molecular machineries which enable them to act in specific intracellular sites and/or in signaling protein complexes.     

Distinct Expression and Localization of Diacylglycerol Kinase Isozymes in Rat Retina

Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGKβ and DGKι localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGKι localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGKβ distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGKϵ is evident in the outer limiting membrane. DGKζ and DGKα localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.     

Role of the N-terminal hydrophobic residues of DGKε in targeting the endoplasmic reticulum

The endoplasmic reticulum (ER), comprised of an interconnected membrane network, is a site of phospholipid and protein synthesis. The diacylglycerol kinase (DGK) enzyme family catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Both of these lipids are known not only to serve as second messengers but also to represent intermediate precursors of lipids of various kinds. The DGK family is targeted to distinct subcellular sites in cDNA-transfected and native cells. Of DGKs, DGKε localizes primarily to the ER, suggesting that this isozyme plays a role in this organelle. Using experiments with various deletion and substitution mutants, this study examined the molecular mechanism of how DGKε is targeted to the ER. Results demonstrate that the N-terminal hydrophobic sequence 20–40 plays a necessary role in targeting of DGKε to the ER. This hydrophobic amino acid segment is predicted to adopt an α-helix structure, in which Leu22, L25, and L29 are present in mutual proximity, forming a hydrophobic patch. When these hydrophobic Leu residues were replaced with hydrophilic amino acid Gln, the mutant fragment designated DGKε (20–40/L22Q,L25Q,L29Q) exhibits diffuse distribution in the cytoplasm. Moreover, full-length DGKε containing these substitutions, DGKε (L22Q,L25Q,L29Q), is shown to distribute diffusely in the cytoplasm. These results indicate that the N-terminal hydrophobic residues play a key role in DGKε targeting to the ER membrane. Functionally, knockdown or deletion of DGKε affects the unfolding protein response pathways, thereby rendering the cells susceptible to apoptosis, to some degree, under ER stress conditions.     

Diacylglycerol kinases: why so many of them?

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.     

Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells

Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.     

Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung

Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.     

Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.

Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.     

Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.     

Myristic acid specifically stabilizes diacylglycerol kinase δ protein in C2C12 skeletal muscle cells

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.     

Identification and characterization of two splice variants of human diacylglycerol kinase eta

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus. The DGK eta 1 mRNA was ubiquitously distributed in various tissues, whereas the DGK eta 2 mRNA was detected only in testis, kidney, and colon. The expression of DGK eta 2 was suppressed by glucocorticoid in contrast to the marked induction of DGK eta 1. DGK eta 2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (delta 1 and delta 2). Interestingly, DGK eta 1 and DGK eta 2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGK eta 1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGK eta 2 exhibited sustained endosomal association. The experiments using DGK eta mutants suggested that the oligomerization of DGK eta 2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGK eta 2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGK eta gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Diacylglycerol kinase ζ: at the crossroads of lipid signaling and protein complex organization

Diacylglycerol (DAG) and phosphatidic acid (PA) are lipids with unique functions as metabolic intermediates, basic membrane constituents, and second-signal components. Diacylglycerol kinases (DGK) regulate the levels of these two lipids, catalyzing the interconversion of one to the other. The DGK family of enzymes is composed of 10 isoforms, grouped into five subfamilies based on the presence of distinct regulatory domains. From its initial characterization as a type IV DGK to the generation of mouse models showing its importance in cardiac dysfunction and immune pathologies, diacylglycerol kinase ζ (DGKζ) has proved an excellent example of the critical role of lipid-metabolizing enzymes in the control of cell responses. Although the mechanism that regulates this enzyme is not well known, many studies demonstrate its subtle regulation and its strategic function in specific signaling and as part of adaptor protein complexes. These data suggest that DGKζ offers new opportunities for therapeutic manipulation of lipid metabolism.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.