The Rat Myosin myr 5 Is a GTPase-activating Protein for Rho In Vivo: Essential Role of Arginine 1695

Abstract

myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expressing the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.

The Tail Domain of Myosin M Catalyses Nucleotide Exchange on Rac1 GTPases and Can Induce Actin‐Driven Surface Protrusions

“…Binding of the motor domain to F-actin was found to be dependent on ATP and on the presence of a phosphatase inhibitor, in sharp contrast with conventional myosin II. Thus MyoM may act not as a true motor but rather serve to anchor the tail in actin-rich regions of the cell, as has been suggested for Myosin IX (6,10). In addition, whereas the genome of D. discoideum does not seem to encode a myosin with a GAP domain, a MyoM homolog might be hidden in the human genome.…”

“…The tail domain of various myosins contains elements including Src homology 3 (SH3) and pleckstrin homology (PH) domains thought to mediate proteinprotein or protein -lipid interactions within signaling cascades. Particularly, the tail of class IX myosins harbors a GTPase activating protein (GAP) domain specific for Rho (6), a member of the family of small GTPases that play key roles in growth factor-regulated cytoskeletal reorganization and cell cycle progression (7 -9). Mammalian cells overexpressing myr5, a rat myosin IX, lose stress fibers and focal contacts and consequently round up (6).…”

“…Particularly, the tail of class IX myosins harbors a GTPase activating protein (GAP) domain specific for Rho (6), a member of the family of small GTPases that play key roles in growth factor-regulated cytoskeletal reorganization and cell cycle progression (7 -9). Mammalian cells overexpressing myr5, a rat myosin IX, lose stress fibers and focal contacts and consequently round up (6). This phenotype correlates well with the effects induced by inactivation of Rho (6,10).…”

How many is enough? exploring the myosin repertoire in the model eukaryoteDictyostelium discoideum

“…Pleckstrin-homology domains are found in myosin X, and might mediate binding to either phosphoinositides or proteins such as the [3-subunit of the trimeric G proteins (58). Other types of domains have been shown to bind proteins involved in signal transduction cascades, including SH3 domains, and a chimaerin-like GTPase-activating domain for a small GTPase of the rho family (59,60).…”

“…Among these, the pleckstrin homology domains of myosin X and the chimaerin-like GAP domain for Rho GTPases of myosin IX are particularly exciting (59,60). In myosin Is, SH3 domains form the TH3 tail domain mentioned above.…”