TY - JOUR
T1 - Structure-function relationships of the mouse Gap1 m. Determination of the inositol 1,3,4,5-tetrakisphosphate-binding domain
AU - Fukuda, Mitsunori
AU - Mikoshiba, Katsuhiko
PY - 1996
Y1 - 1996
N2 - Gap1(IP4BP), one of a member of Ras GTPase-activating proteins, has been identified as a specific inositol 1,3,4,5-tetrakisphosphate (IP 4)-binding protein (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P, and Irvine, R. F. (1995) Nature 386, 527530). In this paper we describe Gap1 m, which is closely related to Gap1(IP4BP), to also be an IP 4-binding protein and show that the pleckstrin homology domain (PH) is the central IP 4-binding domain by expressing fragments of the mouse Gap1 m in Escherichia coli as fusion proteins and examining their activities. However, in addition to the PH domain, an adjacent GAP-related domain and carboxyl terminus are required for high affinity specific IP 4 binding. The PH domain is highly conserved in the Gap1 family and also has striking homology to the amino-terminal region of Bruton's tyrosine kinase. Substitution of Cys for Arg at position 628 in the PH domain corresponding to the mutation of Bruton's tyrosine kinase observed in X-linked immunodeficiency mice results in a dramatic reduction of IP 4 binding activity as well as phospholipid binding capacity of Gap1 m. This mutant also showed the GAP activity against Ha-Ras to be similar to that of the wild type Gap1 m. Our results suggest that the PH domain of Gap1 m functions as a modulatory domain of GAP activity by binding IP 4 and phospholipids.
AB - Gap1(IP4BP), one of a member of Ras GTPase-activating proteins, has been identified as a specific inositol 1,3,4,5-tetrakisphosphate (IP 4)-binding protein (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P, and Irvine, R. F. (1995) Nature 386, 527530). In this paper we describe Gap1 m, which is closely related to Gap1(IP4BP), to also be an IP 4-binding protein and show that the pleckstrin homology domain (PH) is the central IP 4-binding domain by expressing fragments of the mouse Gap1 m in Escherichia coli as fusion proteins and examining their activities. However, in addition to the PH domain, an adjacent GAP-related domain and carboxyl terminus are required for high affinity specific IP 4 binding. The PH domain is highly conserved in the Gap1 family and also has striking homology to the amino-terminal region of Bruton's tyrosine kinase. Substitution of Cys for Arg at position 628 in the PH domain corresponding to the mutation of Bruton's tyrosine kinase observed in X-linked immunodeficiency mice results in a dramatic reduction of IP 4 binding activity as well as phospholipid binding capacity of Gap1 m. This mutant also showed the GAP activity against Ha-Ras to be similar to that of the wild type Gap1 m. Our results suggest that the PH domain of Gap1 m functions as a modulatory domain of GAP activity by binding IP 4 and phospholipids.
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U2 - 10.1074/jbc.271.31.18838
DO - 10.1074/jbc.271.31.18838
M3 - Article
C2 - 8702543
AN - SCOPUS:0029748612
VL - 271
SP - 18838
EP - 18842
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 31
ER -