Abstract
The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.
Highlights
The PAK-I family share an homologous C-terminal catalytic domain and a conserved auto
Binding of Rac to N1 and N4 does not require lipids, which were not present in these assays. Despite their significant sequence differences, the mechanism of regulation of Acanthamoeba MIHCK and mammalian PAK1 are quite similar and, in this respect as well as by sequence, MIHCK more closely resembles the mammalian PAK-I family than the PAK-II family; MIHCK has an autoinhibitory domain
The PAN region of MIHCK is sufficient for inhibition of the activity of full-length MIHCK and the catalytic domain, and inhibition is abolished by autophosphorylation just as for PAK1 [10, 14]
Summary
Construction of Full-length MIHCK cDNA—To construct a fulllength cDNA clone, clones 43 (which lacks 23 bp at the 5Ј end) and 45 (which has a 3-bp deletion, bp 613– 615) [3] were digested with KpnI and XmnI; the large digestion fragment of clone 45 (4.8 kb) was ligated to the small fragment of clone 43 (2 kb) producing plasmid pCB1 containing the complete sequence of MIHCK in pBK-CMV vector. N4 cDNA had 5Ј-BglII and 3Ј-HindIII sites introduced during PCR and was cloned between these sites into pFLAG-2 vector (Sigma) resulting in an N-terminal FLAG sequence in the expressed N4 peptide. Expression of these cDNAs in BL-21(DE3) cells was induced with isopropylthio--Dgalactoside. Assays—Unless otherwise stated, kinase activity was assayed as described previously [24] using synthetic peptide PC9 (200 M), which corresponds to the phosphorylation site of Acanthamoeba myosin IC [29], as substrate at 20 °C in activity buffer containing 50 mM imidazole, pH 7.0, 2.5 mM [␥-32P]ATP (30,000 cpm/nmol), 3.5 mM MgCl2, 0.2 mg/ml bovine serum albumin, 1 mM EGTA. C, expressed MIHCK (37 nM) was dephosphorylated and its activity assayed for 1 min at 20 °C with addition of 100 M PS, 1.5 M GTP-Rac, and 12 M calmodulin (CaM) in the presence of 0.1 mM Ca2ϩ or 2 mM EGTA as shown
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
