The Impact of Meprin A‐Mediated Cleavage on the Function of the Catalytic Subunit of Protein Kinase A (PKA Cβ1)
Authors: Beckett Letitia , Patwardhan Supriya , Delva Jeannette , Jefferson Javon , Watson Melanie , Ongeri Elimelda Moige , Newman Robert H. 
Published: November 2023

The AGC family of protein kinases are serine/threonine kinases that regulate a variety of cell signaling pathways, ranging from growth factor‐mediated regulation of cell cycle and survival to modulation of extracellular matrix metabolism (ECM) in kidney cells. Meprins are zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules. Meprins have been implicated in the progression kidney disease (e.g., ischemia‐reperfusion‐induced acute kidney injury and diabetic nephropathy (DN)). Under certain pathological conditions, meprins are redistributed from the brush‐border membranes to the cytosol and basolateral membranes, where they are able to cleave select proteins. Identified kidney meprin targets include two members of the AGC kinase family, namely the catalytic subunit of protein kinase A (PKA C) and protein kinase C (PKC). Proteolytic processing of PKA C by meprins is isoform specific, both with respect to the PKA C isoform being cleaved and the meprin isoform. Additionally, we found that the beta isoform of PKA C (PKA Cβ1) was differentially cleaved by meprin A and meprin B. While meprin B cleaves PKA Cβ1 at two sites (one between K22/A23 in the N‐terminal tail and another between E322/E323 in the C‐terminal tail), meprin A cleaves PKA Cβ1 at only one site (between R29/K30 in the N‐terminal tail).

The current study demonstrates that this cleavage pattern has functional consequences. For instance, meprin B‐mediated cleavage abolishes PKA Cβ1 activity toward the short peptide substrate, Kemptide, while meprin A‐mediated cleavage does not have any apparent effect on its enzymatic activity. These observations are consistent with both structural and biochemical data that suggest that the C‐terminal tail is involved in forming the ATP binding pocket in PKA C isoforms while the N‐terminal tail is primarily involved in auxiliary functions, such as protein‐protein interactions (e.g., with the scaffold protein, A‐kinase interacting protein (AKIP)). To determine the impact that meprin A‐mediated cleavage has on PKA Cβ1 substrate selection, we used a coupled ATP depletion assay to measure PKA Cβ1 activity toward various substrates. Meprin A‐mediated cleavage did not adversely affect PKA Cβ1's ability to phosphorylate a short peptide substrate (Kemptide). In contrast, pre‐incubation with meprin A significantly reduced PKA Cβ1's activity toward a collection of full‐length native protein substrates, namely histone H3, myelin basic protein and casein. This raises the intriguing possibility that meprin A‐mediated cleavage of the N‐terminal tail may shift the substrate preference of PKA Cβ1 such that distinct subsets of substrates are phosphorylated by the cleaved and uncleaved forms of the kinase. We are investigating this possibility further using functional protein microarrays.

Taken together, these studies suggest that meprin A‐mediated cleavage of PKA Cβ1 at the N‐terminal tail alters its ability to recognize some, but not all, substrates. This may lead to differential phosphorylation profiles in the cleaved and uncleaved states. These studies may offer important insights into meprin‐mediated regulation of PKA‐dependent signaling pathways during chronic and acute kidney injury.

Support or Funding Information

This work was supported by NIH/NIGMS grant 1SC2GM113784‐01 to RHN and NIH/NIGMS grant SC3GM102049‐01 to EMO. LB is supported by the MS‐RISE Program at NC A&T (NIH grant 1R25GM076162‐01A1); JD is supported by the LS‐AMP Bridge to the Doctorate Program at NC A&T; and JJ and MC were supported by the NSF iBLEND Biomathematics Undergraduate Training Program (DBI1038160) to RHN.