Sequence analysis of the full-length cDNA clone showed that gek encodes a large protein of 1613 amino acids (Figure 1a). The N-terminus of Gek contains a predicted Ser/Thr kinase catalytic domain (Figure 1b). This is followed by a large coiled-coil domain with sequence homology to the myosin heavy chain (Figure 1a), a Cys-rich domain similar to the phorbol ester / diglyceride binding structure of protein kinase C Domain (Figure 1c), and a pleckstrin homology domain (Figure 1d), which is present in many signaling molecules for protein-lipid interactions and recruitment to the cell surface. Near the C-terminus of Gek is a sequence similar to the Cdc42/Rac interactive binding (CRIB) domain (Figure 1e). In fact, the deletion of three residues in Gek (GekΔISP) (Figure 1e) corresponds to the three conserved residues in the CRIB domain, disrupting its binding to Dcdc42. Gek shows strong sequence similarity to human tonic dystrophin protein kinase (DMPK). Gek and DMPK share 63% amino acid sequence identity within the 271-aa catalytic core (Figure 1b), and the sequence similarity between the two proteins exceeds the catalytic domain in both directions (Figure 1a and b). However, DMPK is much smaller than Gek. Interestingly, a recently identified class of Rho-binding kinases may function as small GTPase Rho effectors, with domains similar to Gek (Figure 1a). In addition, the kinase domain of Rho-binding kinase is similar to DMPK, although DMPK is more similar to mammalian Rho kinase than Drosophila Gek (identities in the catalytic core are 63% and 49%, respectively). Similarly, the phorbol ester-binding domain of protein kinase C is more similar to Gek's Cys-rich domain (Figure 1c) than Rho-binding kinase.
Figure 1. Primary structure of Gek. ( L, Luo; et al.1997)
Gek is a protein kinase.
To test whether Gek showed kinase activity, scientists transfected Drosophila Schneider cells (S2) with a wild-type Gek expression construct labeled with myc epitopes under the control of the ubiquitous actin promoter. Scientist then immunoprecipitated the Gek protein using an anti-myc antibody. By using histones as substrates, scientists detected kinase activity in a complex of immunoprecipitated cells transfected with myc-Gek, but did not immunoprecipitate from mock-transfected cells. To exclude the possibility that the kinase activity was caused by another kinase closely related to Gek during immunoprecipitation, scientists transfected S2 cells with a myc-tagged mutant Gek construct (A105K), which mutant Mutations with lysine residues in kinases predict domains that are essential for kinase activity. Immunoprecipitated myc-GekA105K did not show any kinase activity above the background, although the expression level of GekA105K protein was comparable to that of wild-type Gek, which was demonstrated by Western blotting with anti-myc antibodies. Because single point mutations in the kinase domain are unlikely to disrupt Gek's binding to other related proteins, scientists concluded that Gek proteins have kinase activity.
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