Faculty Bibliography

Post-translational modification of Ras proteins includes prenylcysteine-directed carboxyl methylation. Because Ras participates in Erk activation by epidermal growth factor (EGF), we tested whether Ras methylation regulates Erk activation. EGF stimulation of Erk was inhibited by AFC (N-acetyl-S-farnesyl-L-cysteine), an inhibitor of methylation, but not AGC (N-acetyl-S-geranyl-L-cysteine), an inactive analog of AFC. AFC inhibited Ras methylation as well as the activation of pathway enzymes between Ras and Erk but did not inhibit EGF receptor phosphorylation, confirming action at the level of Ras. Transient transfection of human prenylcysteine-directed carboxyl methyltransferase increased EGF-stimulated Erk activation. AFC but not AGC inhibited movement of transiently transfected green fluorescent protein-Ras from the cytosol to the plasma membrane of COS-1 cells and depleted green fluorescent protein-Ras from the plasma membrane in stably transfected Madin-Darby canine kidney cells, suggesting that methylation regulates Erk by ensuring proper membrane localization of Ras. However, when COS-1 cells were transfected with Ras complexed to CD8, plasma membrane localization of Ras was unaffected by AFC, yet EGF-stimulated Erk activation was inhibited by AFC. Thus, Ras methylation appears to regulate Erk activation both through the localization of Ras as well as the propagation of Ras-dependent signals

The Hedgehog-Gli signaling pathway is involved in the regulation of the proliferation of precursors in different organs of the normal vertebrate embryo. These cells express Gli1 and may be the target of cancer-causing agents. Many tumor types derived from organs that contain Gli1+ precursors appear to consistently express Gli1, indicating their origin and/or the presence of an active pathway. Inappropriate pathway activation in a variety of precursor cells in model organisms leads to tumor formation while inhibition of the pathway in human tumor cells leads to a decrease in their proliferation. In the brain we have documented the expression of Gli1 in germinative zones, and a variety of brain tumors express GLI1, including medulloblastomas of the cerebellum and a number of gliomas of the cerebral cortex. The requirement for SHH-Gli signaling in the growth of the mouse brain, together with the ability of inappropriate pathway activation in the cerebellum to cause medulloblastomas, and the inhibition of the growth of a number of brain tumors with cyclopamine, a SHH signaling inhibitor, underscores the critical role of the SHH-GLI pathway in brain growth and tumor formation. Moreover, they highlight the components of this pathway as prime targets for drug development, with special emphasis on the GLI proteins. Such reagents would allow a rational therapeutic approach to highly intractable diseases

Recent mutant analysis in zebrafish points to an important role for oriented cell division in cardiac chamber formation and reveals its molecular control by a novel signal from the heart's interior

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.

Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury

Accumulating evidence has indicated that neurotrophin receptor trafficking plays an important role in neurotrophin-mediated signaling in developing as well as mature neurons. However, little is known about the molecular mechanisms and the components of neurotrophin receptor vesicular transport. This article will describe how neurotrophin receptors, Trk and p75 neurotrophin receptor (p75NTR), are intimately involved in the axonal transport process. In particular, the molecules that may direct Trk receptor trafficking in the axon will be discussed. Finally, potential mechanisms by which receptor-containing vesicles link to molecular cytoskeletal motors will be presented

Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases