Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Abstract

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.

Structure, Properties, and Probable Physiological Role of Small Heat Shock Protein with Molecular Mass 20 kD (Hsp20, HspB6)

“…Due to the lack of space, we will not analyze either effects of phosphory lation and dephosphorylation of myosin light chains or the role of caldesmon (or calponin) in regulation of smooth muscle contraction. These problems are thor oughly described in a number of recently published reviews [17,25,30,37,52]. Let us analyze only the effect of Hsp20 on the regulation of smooth muscle contrac tion.…”

Identification of novelin vitroprotein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: nano-liquid chromatography tandem mass spectrometry methodology

“…In this study, seven of the identified phosphorylation sites were not predicted by any of the five web servers. Ser1776 and Ser1779 were earlier identified as PKA sites in smMLCK by Krymsky et al (23) and, later, Khapchaev et al (45) showed that Ser1779 is subject to mitogen-activated protein kinase phosphorylation site and Ser1773 is a PKA phosphorylation site corresponding to nmMLCK. In this study, we were not able to clarify PKA phosphorylation sites on S1776 or S1779 due to a lack of MS 3 data.…”

Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli

“…The polyclonal antibodies to KRP [22] and KRP phosphorylated at Ser 13 and Ser 19 [27] have been described. The anti-rMLC antibody (clone Y-21) and microcystin-LR were from Sigma, USA.…”

“…GST-tagged mitogen-activated protein kinase with a molecular weight of 44 kDa (p44 erkl -MAP kinase) expression and activation by phosphorylation with the constitutively active His 6 -tagged MAPK/ERK (extracellular-signal-regulated kinase) kinase 1 (MEK1) were performed as described [27]. The plasmid pET22-b+/DD-hKRP, expressing the phosphorylation-mimicking KRP mutant, S13D,S19D, was generated using the pET22-b+/hKRP construct encoding the full-length human KRP [27] as a template.…”

“…KRP binds with the acidic C-terminus to the neck region of unphosphorylated myosin, but not to phosphorylated myosin [24], and thereby promotes the extended conformation of myosin and filament formation [25,26]. The flexible N-terminus harbours several phosphorylation sites, including a PKA/PKG (Ser 13 ) and a MAP kinase phosphorylation site (Ser 19 ) [22,[27][28][29][30]. In intact ileum, forskolin leads to a significant phosphorylation of Ser 13 , which correlates with relaxation, whereas phosphorylation of Ser 19 increases only a little in response to PKC mediated activation of MAP kinase [27].…”