Kathleen H. Holt

Desensitization of Ras Activation by a Feedback Disassociation of the SOS-Grb2 Complex

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.

Biosynthesis of dystroglycan: processing of a precursor propeptide

Dystroglycan is a cytoskeleton‐linked extracellular matrix receptor expressed in many cell types. Dystroglycan is composed of α‐ and β‐subunits which are encoded by a single mRNA. Using a heterologous mammalian expression system, we provide the first biochemical evidence of the α/β‐dystroglycan precursor propeptide prior to enzymatic cleavage. This 160 kDa dystroglycan propeptide is processed into α‐ and β‐dystroglycan (120 kDa and 43 kDa, respectively). We also demonstrate that the precursor propeptide is glycosylated and that blockade of asparagine‐linked (N‐linked) glycosylation did not prevent the cleavage of the dystroglycan precursor peptide. However, inhibition of N‐linked glycosylation results in aberrant trafficking of the α‐ and β‐dystroglycan subunits to the plasma membrane. Thus, dystroglycan is synthesized as a precursor propeptide that is post‐translationally cleaved and differentially glycosylated to yield α‐ and β‐dystroglycan.

Functional Rescue of the Sarcoglycan Complex in the BIO 14.6 Hamster Using δ-Sarcoglycan Gene Transfer

Four types of limb-girdle muscular dystrophy (LGMD) are known to be caused by mutations in distinct sarcoglycan genes. The BIO 14.6 hamster is a model for sarcoglycan-deficient LGMD with a deletion in the delta-sarcoglycan (delta-SG) gene. We investigated the function of the sarcoglycan complex and the feasibility of sarcoglycan gene transfer for LGMD using a recombinant delta-SG adenovirus in the BIO 14.6 hamster. We demonstrate extensive long-term expression of delta-sarcoglycan and rescue of the entire sarcoglycan complex, as well as restored stable association of alpha-dystroglycan with the sarcolemma. Importantly, muscle fibers expressing delta-sarcoglycan lack morphological markers of muscular dystrophy and exhibit restored plasma membrane integrity. In summary, the sarcoglycan complex is requisite for the maintenance of sarcolemmal integrity, and primary mutations in individual sarcoglycan components can be corrected in vivo.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Insulin and epidermal growth factor receptors regulate distinct pools of Grb2-SOS in the control of Ras activation.

Insulin and epidermal growth factor (EGF) stimulate a rapid but transient increase in the amount of GTP bound to Ras that returns to the basal GDP-bound state within 10-30 min. Although insulin stimulation resulted in a dissociation of the Grb2·SOS complex, EGF did not affect the Grb2·SOS complex but instead induced dissociation of Grb2-SOS from tyrosine-phosphorylated Shc. The dissociation of Grb2-SOS from Shc was not due to dephosphorylation as Shc remained persistently tyrosine-phosphorylated during this time. Furthermore, there was no decrease in the extent of insulin receptor substrate 1, insulin receptor, or EGF receptor tyrosine phosphorylation. Surprisingly, however, despite the EGF-induced decrease in the amount of Grb2-SOS bound to Shc, the extent of Grb2 associated with Shc remained constant, and there was a concomitant increase in the amount of SOS associated with Grb2. In addition, after the insulin-stimulated dissociation of Grb2 from SOS, EGF treatment induced the reassociation of the Grb2·SOS complex. Quantitative immunoprecipitation demonstrated that only a small fraction of the total cellular pool of Grb2 was associated with SOS. Similarly, only a small fraction of SOS and Grb2 were co-immunoprecipitated with Shc. Together, these data suggest the presence of distinct Grb2-SOS pools that are independently utilized by insulin and EGF in their recruitment to tyrosine-phosphorylated Shc.

Lentiviral Vectors Pseudotyped with Filoviral Glycoproteins

Pseudotyping lentivirus-based vectors is a strategy used to study conferred vector tropism and mechanisms of envelope glycoprotein function. Lentiviruses and filoviruses both assemble at the plasma membrane and have homotrimeric structural envelope glycoproteins that mediate both receptor binding and fusion. Such similarities help foster efficient pseudotyping. Importantly, filovirus glycoprotein pseudotyping of lentiviral vectors allows investigators to study virus entry at substantially less restrictive levels of biosafety containment than that required for wild-type filovirus work (biosafety level-2 vs. biosafety level-4, respectively). Standard lentiviral vector production involves transient transfection of viral component expression plasmids into producer cells, supernatant collection, and centrifuge concentration. Because the envelope glycoprotein expression plasmid is provided in trans, wild type or variant filoviral glycoproteins from marburgvirus or ebolavirus species may be used for pseudotyping and compared side-by-side. In this chapter we discuss the manufacture of pseudotyped lentiviral vector with an emphasis on small-scale laboratory grade production.

Chapter 6 Molecular basis of insulin action

Summary During the past several years, we have made enormous progress in our understanding of the cellular mechanisms involved in insulin receptor signaling. These findings have come about due to the coordinate use of physical, molecular, and cellular biological approaches to the complex issues of intracellular protein-protein interactions, subcellular localization and activation of enzyme activities. Although we have come a long way, we still have a lot to learn before the entire scheme of insulin signaling is established at the molecular level. Presently, there are several pressing issues that need to be resolved in order to determine the basis for the mitogenic and metabolic actions of insulin. For example, one important issue is the molecular basis of receptor signaling specificity. Does this result from the regulation of the signal amplitude (receptor number and/or hormone dose) or from the intrinsic cellular context of a particular receptor. If cell context-dependent, is this due to receptor substrate specificity, site specific phosphorylation, and/or combinational associations within a defined group of effectors. Although some progress is being made examining the potential importance of effector compartmentalization, are the temporal patterns of activation events important? Finally, what are the unidentified factors which may be necessary in the pleiotropic actions of insulin? These issues will only be resolved once each of the biochemical pathways leading to a particular biological response have been defined. We are looking forward to a very exciting future for the elucidation of the molecular basis of insulin action.