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Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

ABSTRACT: Zipper-interacting protein kinase (ZIPK) has been implicated in Ca(2+)-independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC(20) and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC(20) and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N(6) position and an engineered ZIPK capable of utilizing such substrates. (32)P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC(20) as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC(20) was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC(20).

SUBMITTER: Moffat LD

PROVIDER: S-EPMC3196122 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

Moffat Lori D LD Brown Shannon B A SB Grassie Michael E ME Ulke-Lemée Annegret A Williamson Laura M LM Walsh Michael P MP MacDonald Justin A JA

The Journal of biological chemistry 20110831 42

Zipper-interacting protein kinase (ZIPK) has been implicated in Ca(2+)-independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC(20) and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC(20) and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to i ...[more]

PMID: 21880706

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Project description:Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Project description:UnlabelledMammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.ImportancePrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

Project description:Objective: Pupil dilation patterns are outside of conscious control and provide information regarding neuropsychological processes related to deception, cognitive effort, and familiarity. This study examined the incremental utility of pupillometry on the Test of Memory Malingering (TOMM) in classifying individuals with verified traumatic brain injury (TBI), individuals simulating TBI, and healthy comparisons. Method: Participants were 177 adults across three groups: verified TBI (n = 53), feigned cognitive impairment due to TBI (SIM, n = 52), and heathy comparisons (HC, n = 72). Results: Logistic regression and ROC curve analyses identified several pupil indices that discriminated the groups. Pupillometry discriminated best for the comparison of greatest clinical interest, verified TBI versus simulators, adding information beyond traditional accuracy scores. Simulators showed evidence of greater cognitive load than both groups instructed to perform at their best ability (HC and TBI). Additionally, the typically robust phenomenon of dilating to familiar stimuli was relatively diminished among TBI simulators compared to TBI and HC. This finding may reflect competing, interfering effects of cognitive effort that are frequently observed in pupillary reactivity during deception. However, the familiarity effect appeared on nearly half the trials for SIM participants. Among those trials evidencing the familiarity response, selection of the unfamiliar stimulus (i.e., dilation-response inconsistency) was associated with a sizeable increase in likelihood of being a simulator. Conclusions: Taken together, these findings provide strong support for multimethod assessment: adding unique performance assessments such as biometrics to standard accuracy scores. Continued study of pupillometry will enhance the identification of simulators who are not detected by traditional performance validity test scoring metrics. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Dataset Information

Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

Publications

Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

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