Calmodulin-binding and Autoinhibitory Domains ofAcanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

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 MI-HCK 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 fulllength 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. MIHCK1 phosphorylates a single Ser or Thr in the head domain of each of the three class-I myosins from Acanthamoeba castellanii substantially increasing their actin-activated MgATPase activities (for reviews, see Refs. 1 and 2). When the amino acid sequence of its catalytic domain is compared with the catalytic domains of other kinases (3), MIHCK is most similar to PAK1, a member of the PAK-I family of the mammalian Ste20 group kinases (according to the classification system of Dan et al. (4)).The PAK-I family (for reviews, see Refs. 5-8) share an homologous C-terminal catalytic domain and a conserved autoregulatory region (9) in the N-terminal half, also called PAN for PAK N-terminal motif (5, 10). This region is responsible for autoinhibition of mammalian PAK1, -2, and -3 (members of the PAK-I family) and for binding of Rac and Cdc42, which reverses autoinhibition (9 -14). As described by Lei et al. (9), the ϳ80-residue autoregulatory region of mammalian PAK1 consists of an N-terminal p21-binding domain (PBD) of ϳ44 residues, an overlapping inhibitory switch (IS) domain of ϳ50 residues that includes the C-terminal ϳ27 residues of the PBD and a C-terminal ϳ14-residue kinase inhibitory domain. A CRIB (Cdc42/Rac interactive binding) motif (15) of ϳ16 residues near the N terminus of the autoregulatory region is an essential component of the PBD. PAK4 (16) and other PAK-II family members also have a C-terminal catalytic domain and an N-terminal PBD but lack a recognizable autoinhibitory domain (4).Acanthamoeba MIHCK has a C-terminal catalytic domai...

PAK family kinases

“…DdPAKa-null cells display defects in myosin II assembly which delay significantly the onset of chemotaxis 84 . The DdPAKb (MIHCK) is activated by Rac isoforms and acidic lipids and inhibited by Ca 2+ -calmodulin, as also reported for Acanthamoeba MIHCK 85 . In response to chemo-attractant (cAMP) stimulation, 86 PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 sec.…”

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling

“…MIHCK, from Dictyostelium , was validated as PAKb, which performs a similar function to phosphorylate and activate myosin I. AcMIHCK has a homologous region to human PAK1, PBD (Residue 93–149), which includes the CRIB motif (Residue 93–100) and as IS domain, but lacks the kinase inhibitory (KI) domain region. It also has a putative calmodulin-binding region at its N-terminal, before PBD ( Brzeska et al, 2001 ). The typical C-terminal Ser/Thr kinase domain has the characteristic Ser-627 phosphorylation site.…”

Molecular and functional characterization of EhPAK3, a p21 activated kinase from Entamoeba histolytica

“…In ameba, PAKs have been directly implicated in the actin-myosin reorganization. Dictyostelium PAKB (DdMIHCK) and Acanthomeba MIHCK have been shown to directly phosphorylate myosin I heavy chain (Lee et al, 1996;Brezska et al, 2001). These two kinases have in addition a Ca 2+ -calmodulin binding site.…”