ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers

Abstract

Our group recently identified Ras homolog member I (ARHI), a novel maternally imprinted tumor suppressor gene that encodes a 26 kDa GTP-binding protein with high homology to Ras and Rap. Unlike other Ras family members, ARHI exhibits several unusual structural and functional properties. ARHI contains a unique 34 amino-acid extension at the N-terminus, and differs from Ras in residues critical for GTPase activity and in its putative effector domain. Like Ras, ARHI can bind to GTP with high affinity but has low intrinsic GTPase activity. In addition, while Ras is an oncogene, ARHI functions as an inhibitor for cell growth. (32)Phosphorus labeling showed that ARHI is maintained in a constitutively activated GTP-bound state in resting cells, possibly because of impaired GTPase activity. ARHI is associated at the cell membrane through its prenylation at the C-terminal cysteine residue. Mutation of the conserved CAAX box at the C-terminus led to a loss of its membrane association and a decreased ability to inhibit cell growth. Conversion of Ser(51) to Asn decreased GTP binding and reduced ARHI's biological activity. Mutation of Ala(46) to Val increased the ability of ARHI to inhibit cell growth, associated with a further decrease of its intrinsic GTPase activity. Moreover, conversion of residues in ARHI that are conserved in the Ras family for GTPase activity partially restored the GTPase activity in ARHI. Most strikingly, deletion of ARHI's unique N-terminal extension nearly abolished its inhibitory effect on cell growth, suggesting its importance in ARHI's inhibitory function. Thus, ARHI is a unique Ras family member that retains basic small GTPase function, but exhibits many unusual features. In contrast to most other Ras family members, ARHI has a long N-terminal extension, modest GTPase activity, and constitutive GTP binding in resting cells. Furthermore, unlike the Ras oncogene, ARHI inhibits cell growth, and loss of its expression in cells may contribute to the development of breast and ovarian cancers.

The Ras-related protein AGS1/RASD1 suppresses cell growth

“…This activity may be associated with amino acids in the phosphate/magnesium-binding domains (PM1 and PM3) that differ from the corresponding amino acids in H-Ras and K-Ras, mutation of which results in constitutive activation of these proteins. Other Ras-related proteins containing similar amino-acid differences in this region also exhibit constitutive activity (Foster et al, 1996;Luo et al, 2003).…”

“…AGS1/RASD1 falls into a functional grouping with three other Ras-like proteins (RIG, RERG, ARH1/ NOEY2), which were recently shown to suppress cell growth and/or exhibit altered expression in various tumors (Finlin et al, 2001;Ellis et al, 2002;Luo et al, 2003). The AGS1/RASD1 gene maps to human chromosome 17p11.2, a region associated with a high incidence of loss of heterozygosity and deletions in cancers (Soenen et al, 1998;Stacey et al, 1999;Koga et al, 2002).…”

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7

“…3 Downregulation is achieved through multiple mechanisms, including loss of heterozygosity, DNA methylation, transcriptional regulation and shortened RNA half-life. [4][5][6][7][8][9][10][11][12][13][14] ARHI encodes a 26 kDa GTPase with 50-60% homology to Ras and Rap. 5 In contrast to the oncogenic activity of Ras, re-expression of ARHI at physiologic levels slows proliferation, inhibits motility, produces tumor dormancy and induces autophagy.…”

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

“…Here, we show that DiRas3 also binds to activated A-RAF (though with lower affinity) and also inhibits A-RAF:B-RAF heterodimerization, suggesting that A-RAF and C-RAF share the same DiRas3-dependent regulatory mechanism. Knowing that DiRas3 does not bind to the RBD of RAF kinases [7,31], and that the binding of C-RAF and A-RAF to DiRas3 is Ras-dependent, the difference between these two RAF isoforms in their binding affinity for DiRas3 might be a consequence of their difference in binding to Ras. The weak binding of A-RAF to the activated Ras has 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10 been attributed to the presence of a lysine at position 22 of the RBD in A-RAF because exchanging the lysine to an arginine increases the affinity of A-RAF for H-Ras [30].…”