Regulation of the p21-activated Kinase-relatedDictyostelium Myosin I Heavy Chain Kinase by Autophosphorylation, Acidic Phospholipids, and Ca2+-Calmodulin

Lee, Sheu-Fen; Mahasneh, Amjad; Côté, Graham P.

doi:10.1074/jbc.273.43.27911

Cited by 27 publications

(19 citation statements)

References 49 publications

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“…BH search identified strong potential lipid-binding regions located N-terminal to the CRIB domains in all three PAKs (Fig. 6), consistent with localizations previously suggested (40,41,43). Interestingly, BH search also identified strong potential lipid-binding sites N-terminal to CRIB domains in Drosophila PAK A (NP_731073) and C. elegans PAK A (AAA68805).…”

Section: Application Of Bh Search To Proteins With Undetermined Membrsupporting

confidence: 67%

“…This may just be coincidence resulting from an affinity of calmodulin for sites composed of basic and hydrophobic amino acids, but it suggests the possibility that IQ domains may also function as membrane-binding sites, possibly regulated by competition with calmodulin or calmodulin-like proteins. Calmodulin-binding and lipid-binding sites are known to overlap in several proteins (37) including class I myosins (50) and PAKs (40,42), and in some cases, binding of lipid and calmodulin to overlapping sites is mutually exclusive, for example, myristoylated alanine-rich C kinase substrate (37), Acanthamoeba PAK (42) and Dictyostelium PAK (40). IQ domains and other calmodulin-binding sites are usually ␣-helical, and binding of lipids to ␣-helices has been predicted by others (51).…”

Section: Discussionmentioning

confidence: 99%

“…BH searches did not find any potential membrane-binding sites in the tail of striated muscle myosin II. We also performed BH searches on three PAK kinases: Acanthamoeba PAK (AAD11799) and Dictyostelium PAK (AAC71063), both of which are myosin I heavy chain kinases (39,40) and mammalian PAK1 (P35465). All three PAKs bind acidic phospholipids (39 -44), and Acanthamoeba PAK binds to plasma membranes in vivo and in vitro (45).…”

Section: Application Of Bh Search To Proteins With Undetermined Membrmentioning

confidence: 99%

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An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

Brzeska

Guag

Remmert

et al. 2010

Journal of Biological Chemistry

107

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Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg-and Lyspositive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membranebinding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.Recently, there has been considerable interest in characterizing protein domains, such as PH, FERM, C2, and ENTH, that are responsible for specific binding to membrane lipids (for review, see Ref. 1). These domains have highly defined tertiary structures comprising ␣-helices, ␤-sheets and loops, and there are multiple programs for recognizing them in protein sequences. However, a growing number of membrane-binding proteins, including cytoskeletal proteins (2), GTP-binding proteins (e.g. Rit), and GTP-binding protein effectors (e.g. some PAKs) (see references below), do not fit within these categories and bind membrane lipids through much less structured regions. Here, we describe the development and testing of a simple computer search program that identifies such potential membrane-binding sites. This novel search program evolved from our previous effort (3) to identify the membrane-binding site in the heavy chain of Acanthamoeba myosin IC (AMIC) 2 (4), which was known to bind to acidic phospholipids (3, 5, 6) and cell membranes (7-10) through its 220-residue basic region (5).We had found (3) that AMIC binds nonspecifically to acidic phospholipid vesicles in proportion to their negative charge. Prompted by the report (11) that several proteins bind to acidic phospholipids through a basic-hydrophobic-basic (BHB) region consisting of two small clusters of basic amino acids separated by hydrophobic residues, we identified, by visual inspection, a 13-residue BHB sequence, KVKPFLYVLKRR, within the basic region of the AMIC heavy chain (3). A synthetic ...

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Section: Application Of Bh Search To Proteins With Undetermined Membrsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Application Of Bh Search To Proteins With Undetermined Membrmentioning

confidence: 99%

See 1 more Smart Citation

An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

Brzeska

Guag

Remmert

et al. 2010

Journal of Biological Chemistry

107

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“…As in the case of Ste20 in budding yeast, Dictyostelium MIHCK phosphorylates and regulates the activity of myosin. MIHCK might thus link small GTPase signaling pathways to motile processes requiring myosin I molecules (Brzeska et al, 1997;Lee et al, 1998). However, a direct test of this model (e.g.…”

Section: Mihckmentioning

confidence: 99%

The genetics of Pak

Hofmann

Шепелев

Chernoff

2004

Journal of Cell Science

224

234

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p21-activated kinases (Paks) are a highly conserved family of enzymes that bind to and are activated by small GTPases of the Cdc42 and Rac families. With the notable exception of plants, nearly all eukaryotes encode one or more Pak genes, indicating an ancient origin and important function for this family of enzymes. Genetic approaches in many different experimental systems, ranging from yeast to mice, are beginning to decipher the different functions of Paks. Although some of these functions are unique to a given organism, certain common themes have emerged, such as the activation of mitogen-activated protein kinase (MAPK) cascades and the regulation of cytoskeletal structure through effects on the actin and tubulin cytoskeletons.

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“…41 and of MIHCK from Ref. 3. stimulated activation of Dictyostelium MIHCK (23). The probable explanation of this difference is that, unlike Acanthamoeba MIHCK, Rac activation of Dictyostelium MIHCK does not require lipids, and calmodulin inhibits Acanthamoeba MIHCK by competing with lipid binding.…”

Section: Table I Characterization Of the Activity Of Expressed Acanthmentioning

confidence: 98%

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

Brzeska

Young

Tan

et al. 2001

Journal of Biological Chemistry

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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...

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Regulation of the p21-activated Kinase-relatedDictyostelium Myosin I Heavy Chain Kinase by Autophosphorylation, Acidic Phospholipids, and Ca2+-Calmodulin

Abstract: The Dictyostelium myosin I heavy chain kinase (MI-HCK) is a member of the p21-activated kinase family (Lee, S.-F., Egelhoff, T. T., Mahasneh, A., and Cô té , G. P.

Cited by 27 publications

References 49 publications

An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

The genetics of Pak

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

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