Primary Substrate Specificity Determinants for the H4‐Specific Protease‐Activated Protein Phosphotransferase

Eckols, T. Kris; Thompson, Richard E.; Masaracchia, Ruthann A.

doi:10.1111/j.1432-1033.1983.tb07558.x

Cited by 23 publications

(13 citation statements)

References 32 publications

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“…Also of note is that the sequence GTES in sequencing rounds 8 -11 does not occur at any other position in MLCK, further strengthening the conclusion that the phosphorylated residue at this phosphorylation site is Ser-439. The sequences at both phosphorylation sites are consistent with the specificity determinants identified in previous studies (27)(28)(29). Phosphorylation of synthetic peptides from the PAK substrates H4 (27), S6 (28), and RLC (10,29) has demonstrated that optimum reactivity is observed with Arg at position P-1 or P-3.…”

Section: Pak2supporting

confidence: 88%

Phosphorylation of Myosin Light Chain Kinase by p21-activated Kinase PAK2

Goeckeler¹,

Masaracchia²,

Zeng³

et al. 2000

Journal of Biological Chemistry

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138

124

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Phosphorylation of myosin II regulatory light chains (RLC) by Ca2؉ /calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Posttranslational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPasedependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 ؎ 0.21 mol of PO 4 /mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.The PAK 1 family of serine/threonine protein kinases have been implicated in a broad spectrum of signal transduction pathways leading to diverse physiological end points, including cytoskeletal reorganization, apoptosis, and Ras-mediated cell transformation (1, 2). All PAK isoforms are direct effectors of the Rho family GTP-binding proteins Rac and Cdc42, suggesting that cell-specific responses arise from either selective regulation of G protein activation in response to unique agonists or selective phosphorylation of tissue-specific protein kinase substrates.In the initial studies of the relative roles of G protein activation and protein kinase activity in actin reorganization, Sells et al. (3) demonstrated that microinjection of activated PAK1 induced rapid formation of polarized filopodia in Swiss 3T3 cells. However, the relative importance of PAK-dependent phosphorylation of unique substrates during Cdc42/Racdependent cytoskeletal reorganization is incompletely understood. Transient transfection of HeLa cells with constitutively active PAK1 promoted cytoskeletal rearrangement, which was entirely analogous to that observed in Cdc42 and Rac transfected cells (4). In subsequent studies, a peptide that inhibited kinase activity in PAK blocked Cdc42, Rac, and constitutively active PAK-induced morphological changes in transfected HeLa cells (5). An important role for PAK-mediated phosphorylation in actin reorganization has also been supported by studies in permeabilized (6) and intact (7) endothelial cells as well as skinned smooth muscle cells (8). Permeabilized endothelial cells incubated with Cdc42-activated PAK2 or the catalytic domain of PAK2 underwent ATP-dependent retraction and actin reorganization (6). In addition, these studies using purified enzymes established that the regulatory nonmuscle and smooth muscle myosin II light chain is a substrate for PAK2 (9, 1...

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Section: Pak2supporting

confidence: 88%

Phosphorylation of Myosin Light Chain Kinase by p21-activated Kinase PAK2

Goeckeler¹,

Masaracchia²,

Zeng³

et al. 2000

Journal of Biological Chemistry

Self Cite

138

124

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“…15), there is an arginine located two amino acids upstream of this serine. Such a configuration resembles a diagnostic feature of at least two protein kinase target sites (27,28). As appropriate upstream arginines are associated with relatively few of the serines encoded within per ( Fig.…”

Section: Discussionmentioning

confidence: 85%

Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster.

Yu¹,

Jacquier²,

Citri³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

136

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The pero1 and the pers mutations in Drosophila melanogaster, which seem to eliminate or speed up, respectively, the clocks underlying biological rhythmicity, were mapped to single nucleotides. Chimeric DNA fragments consisting of well-defined wild-type plus mutant DNA subsegments were constructed, introduced into flies by germ-line transformation, and assayed for biological activity. These experiments localized both perol and pers to a 1.7-kilobase DNA fragment that is mostly coding DNA. Sequencing of this subsegment from each mutant showed that per"1 is completely accounted for by a nonsense mutation in the third coding exon of a 4.5-kilobase RNA transcribed from this locus. The pers mutation is also a single nucleotide substitution, in the fourth coding exon, which results in a serine-to-asparagine substitution in the per gene protein product. The functional significance of these changes is discussed with reference to the phenotypes of the two mutations.The period gene of Drosophila melanogaster is defined by some of the most interesting behavioral mutations isolated to date. These mutations affect biological rhythms either by eliminating periodic eclosion or behavior or by causing dramatic changes in rhythmic phenotypes (for review see refs. 1 and 2). The original per mutations were induced by Konopka and Benzer (3). Two of the mutants were per' and perL, isolated with respect to circadian eclosion rhythms whose periods are about 5 hr shorter or longer, respectively, than the normal 24 hr. The per' mutant was also recovered (3), based on aperiodic eclosion. This mutation is now called per"l, because there are currently four independently isolated "arrhythmic" mutations at this locus (refs. 4 and 5; R. J. Konopka and M.H., unpublished results).Detailed genetic and phenotypic analyses of these "clock" mutants suggested that per'1 may be a null mutation, behaving like a deletion of this apparently nonvital locus within segment 3B1-2 of the X chromosome (6, 7). Manipulations of the dosage of the wild-type allele and the pers allele suggested that the increased per activity seemingly associated with pers is caused by a qualitative change in the activity of the gene product (4,8).A 6-kilobase (kb) subset of the per region (9, 10) appears to be necessary for per' activity-i.e., rescues per'-induced arrhythmicity of (i) circadian locomotor activity (5, 11, 12), (ii) eclosion (12), and (iii) variations in the male courtship "singing" behavior (5, 12). This portion of the per region corresponds to a 4.5-kb RNA species (9, 10), allowing one to focus on its informational content (13-15).The results we present here include a determination of the precise sites of the mutations in the original arrhythmic and short-period variants. These experiments were undertaken to address some of the issues raised above and the role of the gene in the development (e.g., refs. 16 and 17) and the function (e.g., refs. 15 and 18) of biological clocks in Drosophila. Thus, we set out to determine ifperol is in fact a null mutant and t...

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“…Alternatively, site 3 may represent an alternate trypsin cleavage of site 1. Previous studies have demonstrated that the S6/H4 kinase requires a dibasic sequence, preferably K-R, amino-terminal to the modified serine (25). Depending on the enzyme/trypsin ratios and extent of endogenous phosphorylation, site 3 may be generated by trypsin cleavage between the K and R, as opposed to cleavage at the carboxyl side of R for site 1.…”

Section: Discussionmentioning

confidence: 99%