Myosin I heavy chain kinase: cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42

Abstract

Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy chains of amoeba myosins I, increasing their actin-activated ATPase activities. The activity of MIHCK is increased by binding to acidic phospholipids or membranes and by autophosphorylation at multiple sites. Phosphorylation at a single site is necessary and sufficient for full activation of the expressed catalytic domain. The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain. By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-length genomic DNA and cDNA of MIHCK and have shown it to contain the conserved p21-binding site common to many members of the PAK family. Like some mammalian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GTP-dependent and accompanied by autophosphorylation. In contrast to mammalian PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidic lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids. The acidic lipid-binding site is near the N terminus followed by the p21-binding region. The N-terminal regulatory domain of MIHCK contains alternating strongly positive and strongly negative regions. and the extremely Pro-rich middle region of MIHCK has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain.

Figure 1

Schematic illustration of cloning of Acanthamoeba MIHCK genomic DNA and cDNA. Genomic DNA was cut with XbaI and the digestion mixture was probed with probe A. A genomic DNA library constructed from the positive band was screened with probe A, and one of the positive clones (4.5 kb) was fully sequenced. A cDNA library was constructed by using poly(A) primer (primer C) and two gene-specific primers (primer B and primer A), screened with probes N and C, and 2 of over 50 positive clones were fully sequenced, and 2 were partially sequenced. For further details see Materials and Methods. The numbering in the figure corresponds to the cDNA beginning at the first coding ATG. The positions and sizes of the introns in the genomic DNA are marked. The diagonal lines define the previously cloned catalytic domain. The complete genomic and cDNA sequences are available in GenBank under accession numbers AF104910 and U67056, respectively.

Figure 2

Deduced protein sequence of MIHCK. The C-terminal catalytic domain and the p21-binding region near the N terminus are boxed; the CRIB motif is underlined; acidic residues are in red, basic in green, and prolines in blue. Residues 451–753 are identical to the sequence of the previously cloned catalytic domain (13) and sequences 102–117, 198–212, 255–262, 285–299, 314–328, 437–462, and 626–640 correspond to the N-terminal sequences of seven proteolytic fragments of native kinase (11, 12).

Figure 3

Schematic representation of the MIHCK molecule. The numbers correspond to the residue numbers in full-length kinase. The regions of interest discussed in the text are indicated at the top of the figure. In addition to the essential autophosphorylation site (P) shown in the catalytic domain, there are multiple other autophosphorylation sites (P), especially in the central, basic, Pro-rich region (ref. ; and see text) and possibly in the N-terminal region (?). The origins of the proteolytic fragments described previously (11, 12), and whose properties are discussed in the text, are shown at the bottom of the figure.

Figure 4

(A) Comparison of the PBDs of MIHCK and α-PAK. The CRIB region (27) is marked at the top of the figure. The conserved residues from comparisons of the sequences of many p21-binding proteins (28, 30, 33) are in bold at the top of the figure and, when present, also in bold in the MIHCK and PAK sequences. (B) Comparison of regions of high sequence identity in the Pro, Gly, Ala-rich regions of MIHCK and myosin IC.

Figure 5

Activation of MIHCK by Rac and Cdc42. (A) Phosphatidylserine-dependent activation of unphosphorylated MIHCK by Rac. MIHCK was incubated for 1 min at 20°C with synthetic peptide PC9 in the absence or presence of phosphatidylserine, at the indicated concentrations, and in the absence (open bars) or presence (closed bars) of GST–Rac–GTP[γS]. The fold-activation by Rac is shown at the top of the bars. (B) Autophosphorylation of MIHCK under identical conditions as in A. (C) Effect of Rac on the activity of phosphorylated MIHCK. MIHCK was fully autophosphorylated by incubation with ATP in the absence of phosphatidylserine and then assayed for 1 min at 20°C in the absence or presence of 500 μM phosphatidylserine and in the absence or presence of GST–Rac–GTP[γS], as indicated. (D) GTP[γS] dependence of activation of MIHCK by Rac and Cdc42. Unphosphorylated MIHCK was assayed in the presence of 500 mM phosphatidylserine and in the absence or presence of Rac, Cdc42, and Rho. All GTP-binding proteins were His-tagged at the N terminus and either in GTP[γS] or in GDP-bound form, as indicated in the figure. Activity was measured at 20°C for 1 min with Cdc42 and Rho and for 2 min with Rac. All values were normalized to the activity of MIHCK in the absence of p21s.

Figure 6

Effect of various lipids on the activity of unphosphorylated MIHCK with and without Rac. (A) The activity of unphosphorylated MIHCK was assayed at 30°C in the absence (open bars) or presence of 50 μM (crossed bars) or 500 μM (black bars) phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), linoleic acid (LA), oleic acid (OA), sphingosine (SPH), or ceramide (CER). The cross-hatched bar (PK) shows the activity of fully phosphorylated (maximally activated) MIHCK under the same conditions. (B) Activation of unphosphorylated MIHCK by Rac in the presence of various lipids. Activity was measured for 1 min at 20°C in the presence of phosphatidylserine (PS), phosphatidylinisitol (PI), phosphatidic acid (PA), or linoleic acid (LA) at the indicated concentrations in the absence (hatched bars) or presence (solid bars) of GST–Rac–GTP[γS]. The open bar (K) shows the activity of unphosphorylated MIHCK in the absence of lipid and Rac.