Biomedical subjects

M A Booden

Publications and source records attributed to M A Booden.

6 recordsLinked to original sources

PubMed2001

Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH(2)-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.

Cell Transformation, Neoplastic↗

PubMed2001

Targeting proteins to membranes, using signal sequences for lipid modifications.

Changing an existing lipid or appending a lipid to a cytosolic protein has emerged as an important technique for targeting proteins to membranes and for constitutively activating the membrane-bound protein. The potential for more precise or regulated interactions of lipidated proteins in membrane subdomains suggests that this method for membrane targeting will be of increasing usefulness.

Amino Acid Sequence↗

PubMed2000

Mutation of Ha-Ras C terminus changes effector pathway utilization.

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.

Actins↗

PubMed2000

S-Nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells.

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.

3T3 Cells↗

PubMed1999

Transient palmitoylation supports H-Ras membrane binding but only partial biological activity.

H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.

3T3 Cells↗

PubMed1999

A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation.

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins.

3T3 Cells↗