Exploring the functional role of Nlrp12's interaction with Hck

Abstract

The members of the family of Nucleotide‐binding oligomerization domain‐Like Receptor proteins (NLR) are composed of a N‐terminal domain, a central nucleotide‐binding domain, and a C‐terminal ligand‐binding domain. They are generally known to be involved in regulating NF‐kB pathway. A yeast two‐hybrid screen was performed with the pyrin plus nucleotide binding domain of NLR family pyrin domain‐containing12 protein (Nlrp12) as a bait and human leucocyte proteins a prey. It revealed that Hck, a src non‐receptor tyrosine kinase family member was the top hit. Further experiments confirmed that: 1) the C‐terminal 40 amino acids of Hck specifically binds to Nlrp12's pyrin plus nucleotide binding domains; 2) the last 30 amino acids of Hck is sufficient to bind to Nlrp12's pyrin plus nucleotide binding domains, but neither to Nlrp12's pyrin domain alone nor Nlrp12's nucleotide‐binding domain alone; and, 3) the last 40 amino acids of dephosphorylated, but not phosphorylated, Hck preferentially binds to full‐length Nlrp12. Nlrp12 co‐immunoprecipitated with Hck when both are heterologously overexpressed in 293T cells. Using the same system, Nlrp12 and Hck are co‐localized by immunofluorescence. In addition, in Raw 264.7, U937, and THP‐1 cells, endogenous Hck co‐immunoprecipitated with transfected Nlrp12. Since Csk is a tyrosine kinase of members of the src family that specifically binds to the tyrosine negative regulatory residue at the C‐terminal of src family kinases and phosphorylates Hck to inactivate it in mammalian cells, it is hypothesized that Nlrp12 competes with Csk binding to Hck, thereby activating Hck. Since Nlrp12 also inhibits the NF‐kB pathway, an alternative hypothesis is that Hck rescues cells from Nlrp12's inhibition of NF‐kB activity. An NF‐kB luciferase assay confirmed that overexpression of Hck alone does not inhibit NF‐kB, but overexpression of Hck plus Nlrp12 rescued Nlrp12's inhibition of the NF‐kB pathway.Support or Funding InformationUSC School of Pharmacy Mutual Funds 2017–2019This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.