A Conserved Dimer and Global Conformational Changes in the Structure of apo-PknE Ser/Thr Protein Kinase from Mycobacterium tuberculosis

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

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Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00112.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. G361-G370 ◽

Cited By ~ 10

Author(s):

Eikichi Ihara ◽

Lori Moffat ◽

Meredith A. Borman ◽

Jennifer E. Amon ◽

Michael P. Walsh ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Conformational Changes ◽

Kinase Inhibitor ◽

Smooth Muscles ◽

Dominant Negative ◽

Myosin Phosphatase ◽

Interacting Protein ◽

Zipper Interacting Protein Kinase

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

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