Faculty Bibliography

Formation of the embryonic heart tube requires the medial migration and merger of bilateral precursor populations. A new study of zebrafish cardiogenesis published in this issue of Developmental Cell indicates that precursor migration involves formation of a coherent epithelium and that fibronectin plays an important role in maintaining cardiac epithelial integrity

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function

Small heat shock protein is a ubiquitous molecular chaperone, which consists of a non-conserved N-terminal region followed by a conserved alpha-crystallin domain. To understand the role of the N-terminal region, we constructed N-terminal truncation mutants of StHsp14.0, the sHsp from Sulfolobus tokodaii strain 7. All the mutants formed a stable oligomeric complex similar to that of the wild type. Electron microscopy and size exclusion chromatography-multiangle light scattering showed that the N-terminal region should locate in the center of the oligomeric particle. The mutants exhibited reduced chaperone activity for the protection of 3-isopropylmalate dehydrogenase from thermal aggregation. This reduction correlates with lowered subunit exchange efficiency. The oligomeric structure was retained even after incubation at 90 degrees C. These results suggest that the N-terminal region of StHsp14.0 functions in the thermally induced disassembly of the complex.

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12 A by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity

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