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Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

ABSTRACT: The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity.We used immunofluorescence and Western blotting to show that Ack1 deletion mutants lacking the N-terminus displayed significantly reduced autophosphorylation in cells. A minimal construct comprising the N-terminus and kinase domain (NKD) was autophosphorylated, while the kinase domain alone (KD) was not. When expressed in mammalian cells, NKD localized to the plasma membrane, while KD showed a more diffuse cytosolic localization. Co-immunoprecipitation experiments showed a stronger interaction between full length Ack1 and NKD than between full length Ack1 and KD, indicating that the N-terminus was important for Ack1 dimerization. Increasing the local concentration of purified Ack1 kinase domain at the surface of lipid vesicles stimulated autophosphorylation and catalytic activity, consistent with a requirement for dimerization and trans-phosphorylation for activity.Collectively, the data suggest that the N-terminus of Ack1 promotes membrane localization and dimerization to allow for autophosphorylation.

SUBMITTER: Prieto-Echague V

PROVIDER: S-EPMC2987765 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

Prieto-Echagüe Victoria V Gucwa Azad A Brown Deborah A DA Miller W Todd WT

BMC biochemistry 20101027

<h4>Background</h4>The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity.<h4>Results</h4>We used immunofluorescence and Western blotting to ...[more]

PMID: 20979614

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Project description:The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen Helicobacter pylori. Secreted H. pylori HtrA (HtrA Hp ) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrA Hp forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrA Hp , indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrA Hp from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in H. pylori. We analyzed the role of the amino-terminal cleavage sites on activity, secretion and function of HtrA Hp . Three-dimensional modeling suggested a trimeric structure and a role of amino-terminal processing in oligomerization and regulation of proteolytic activity of HtrA Hp . Furthermore, point and deletion mutants of these processing sites were generated in the recently reported Campylobacter jejuni ?htrA/htrAHp genetic complementation system and the minimal sequence requirements for processing were determined. Polarized Caco-2 epithelial cells were infected with these strains and analyzed by immunofluorescence microscopy. The results indicated that HtrA Hp processing strongly affected the ability of the protease to disrupt the E-cadherin-based cell-to-cell junctions. Casein zymography confirmed that the amino-terminal region is required for maintaining the proteolytic activity of HtrA Hp . Furthermore, we demonstrated that this cleavage influences the secretion of HtrA Hp in the extracellular space as an important prerequisite for its virulence activity. Taken together, our data demonstrate that amino-terminal cleavage of HtrA Hp is conserved in this pathogen and affects oligomerization and thus, secretion and regulatory activities, suggesting an important role in the pathogenesis of H. pylori.

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Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

Publications

Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

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