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R B Pearson

Publications and source records attributed to R B Pearson.

43 records · Page 3Linked to original sources
PubMed1983

Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.

The substrate specificity of the chicken gizzard myosin light chain kinase has been studied by using a series of synthetic peptide analogs of the NH2-terminal sequence of the chicken gizzard myosin light chain (Mr = 20,000). An 18-residue synthetic peptide, (sequence in text) corresponding to the sequence reported by Maita et al. [Maita, T., Chen, J. I. & Matsuda, G. (1981) Eur. J. Biochem. 117, 417-424], was phosphorylated with a 22-fold higher Km and a Vmax that was decreased to 1% of the native protein substrate. This peptide was also an inferior substrate when compared with an 18-residue synthetic peptide with an alternative sequence, (sequence: see text) which was phosphorylated with an apparent Km of 6.9 microM, comparable to the native protein substrate of 8.6 microM, and a Vmax of 3.9 mumol X min-1 X mg-1, 11% of that for the protein substrate. The kinetic of phosphorylation of shortened peptides corresponding to both sequences, together with peptides with appropriate substitutions, indicated that basic residues were the primary determinants of specificity for the smooth muscle myosin light chain kinase. In the latter peptide sequence, lysine residues 11 and 12 and the arginine at position 13 had a major influence on the kinetics of peptide phosphorylation.

Amino Acid Sequence↗
PubMed1982

Phosphorylation of a synthetic heptadecapeptide by smooth muscle myosin light chain kinase.

A synthetic heptadecapeptide corresponding to part of the NH2-terminal 17 residues of chicken gizzard myosin light chain (Mr = 20,000), Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-(P)-Asn-Val-Phe-Ser-NH2, was readily phosphorylated by the myosin light chain kinase isolated from the same tissue. The synthetic peptide was phosphorylated stoichiometrically at serine 13, the same residue phosphorylated in the parent protein. The apparent Km and Vmax for peptide phosphorylation was 90 microM and 1.3 mumol min-1 mg-1 compared to 10 microM and 22 mumol min-1 mg-1, respectively, for the myosin light chain. The synthetic heptadecapeptide acted as a competitive inhibitor for myosin light chain phosphorylation with Ki approximately 600 microM. Acetylation of the heptadecapeptide alpha-amino group of serine 1 had little effect on Vmax (0.8 mumol min-1 mg-1) and increased the apparent Km 2-fold. The smooth muscle myosin light chain kinase did not phosphorylate the synthetic heptadecapeptide analog of the corresponding skeletal muscle myosin light chain (Mr = 18,500), nor did it phosphorylate synthetic peptide substrates specific for the cAMP-dependent protein kinase or phosphorylase b kinase. These findings support the idea that the myosin light chain kinase has particular protein substrate specificity requirements and that some of these are derived from the region of primary structure around the phosphorylation site in its native substrate.

Amino Acid Sequence↗
PubMedDate not supplied

The Akt kinase signals directly to endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Amino Acid Sequence↗
PubMedDate not supplied

Myosin light chain kinase autoregulatory pseudosubstrate prototope.

The myosin light chain kinase is catalytically inactive unless activated by calmodulin. An autoregulatory pseudosubstrate region located on the carboxyl-terminal side of the enzyme's catalytic domain is responsible for maintaining the enzyme in a latent form. This pseudosubstrate region overlaps the calmodulin binding domain. Synthetic peptides corresponding to the regulatory region can have both substrate antagonist and calmodulin antagonist activities. The pseudosubstrate peptide from the smooth muscle myosin light chain kinase, smMLCK(787-807), S787KDRMKKYMARRKW800QKTGHAV807 is a potent substrate antagonist with a Ki of approximately 12 nM and acts as a calmodulin antagonist with an IC50 = 0.54 microM. The shorter peptide R797RKWQK802, Ki = 1.26 microM, is the core region primarily responsible for substrate antagonist activity and is a weak calmodulin antagonist, IC50 = 181 microM. The corresponding skeletal muscle peptide, KRRWKK was a comparable substrate antagonist, IC50 = 1.63 microM, but a 30-fold more potent calmodulin antagonist, IC50 = 6.1 microM. Substitution of the Trp in either peptide with Phe or Leu did not significantly alter the substrate antagonist activity but markedly reduced calmodulin antagonist activity, RRKWQK, IC50(calmodulin) = 181 microM; RRKFQK, IC50(calmodulin) = 488 microM; RRKLQK, IC50(calmodulin) = 1700 microM; KRRWKK, IC50(calmodulin) = 6.1 microM; KRRLKK, IC50(calmodulin) = 221 microM and KRRFKK, IC50(calmodulin) = 93 microM. The IC50(substrate) values for these peptides ranged from 0.5-13 microM. The peptide KRRLKK was the most selective substrate antagonist and is suitable as an inhibitor for the myosin light chain kinase with the ratio IC50(calmodulin): IC50(substrate) = 273 and an IC50(substrate) = 0.81 microM.

Adenosine Triphosphate↗