||Regulator of G protein signalling (RGS) proteins negatively regulate G protein signalling via interacting with and accelerating the intrinsic GTPase activity of the activated Gα subunits. Among 30 mammalian RGS proteins, Gα-interacting protein (GAIP) belongs to the A/RZ subfamily. GAIP has a simple structure composed of a highly conserved RGS domain flanked by an amino-terminal cysteine string motif and a short-carboxyl extension. GAIP is a phospho-protein, with eight phosphorylation sites distributed among all three regions. It also has different binding motifs for palmitolyation and interactions with adapter proteins. There is evidence to suggest that GAIP may play a role in the modulation of the extracellular signal-regulated protein kinase (ERK) pathway. The objective of the present study was to investigate the functional role of GAIP in the regulation of other mitogen-activated protein kinase (MAPK) pathways. In HEK293 cells, over-expression of GAIP impaired serum-induced stimulation of the ERK, JNK and p38 MAPK pathways. Using various mutants of the upstream regulators of MAPKs, the site of action of GAIP was mapped to the level at or above the monomeric small G proteins such as Ras, Rac and Cdc42. Serine-to-alanine mutation at site 151 and C-terminal truncation of GAIP restored the serum-induced activations of MAPK pathways. The suppressions of serum-induced activations of Ras and ERK were abolished by U0126 (a MEK inhibitor) and over-expression of Gαi3Q204L. These findings indicated that ERK phosphorylation Ser151, PDZ binding motif, ERK phosphorylation and protein-protein interaction are probably engaged in transducing the effect of GAIP on MAPK pathways. On the other hand, Ser151 plays a role in the cell proliferation in HEK293 cells and in the suppression of Ras-mediated cell transformation (including foci formation and anchorage independent growth in soft agar) in NIH/3T3 cells. In conclusion, these results provide indications that GAIP serves as a negative regulator of serum-induced activations of MAPK pathways.