In this study, we sought to investigate the signaling pathways through which Rho regulates apical junction formation in bronchial epithelial cells. A library of SMARTpool siRNAs targeting 28 Rho target proteins was screened in 16HBE cells, and the protein kinase PRK2 identified. PRK2 belongs to a family of 3 serine-threonine kinases, the PKC-related kinase family, also called PKN (protein kinase novel) (24
). PRK isoforms show homology to PKC family kinases within their conserved C-terminal kinase domains and contain an N-terminal GTPase-binding domain and a central domain with weak homology to the calcium-dependent phospholipid binding C2 domain of PKC (Fig. ). The GTPase-binding domain, also called the HR1 (homology region 1), contains 3 tandem repeats of approximately 70 amino acids encoding antiparallel coiled-coil domains (referred to as HR1a to -c), which form independent GTPase-binding modules. In the case of PRK1 at least, only HR1a and HR1b bind to Rho GTPases (11
). Initial studies of PRK1 found it to interact with active RhoA, RhoB, and RhoC but not Rac1, and when PRK2 was cloned, a similar specificity for Rho but not Rac was observed (3
). PRK2 has since been shown to interact with Rac, as well as Rho (41
); however, we found only a very weak interaction between PRK2 and a constitutively active mutant of Rac1 in coimmunoprecipitation experiments. Point mutations were introduced into the HR1a and HR1b domains to prevent binding of PRK2 to active RhoA, and this mutant failed to rescue apical junction formation when endogenous PRK2 was depleted, showing that PRK2 acts as a RhoA target during apical junction formation.
A single PRK/PKN homolog exists in Drosophila
, and there is evidence that it regulates epithelial morphogenesis. Null mutants of Drosophila
PKN show defects in dorsal closure, a developmental process in which leading-edge cells of the epidermis elongate along the dorsal-ventral axis until they meet at the dorsal midline (20
). Dorsal closure requires dynamic regulation of cell-cell adhesion and actomyosin-dependent cell shape changes, and PKN has been proposed to regulate this downstream of Rho (5
). Biological roles for mammalian PRK proteins have not been clearly elucidated; however, overexpression studies have provided some clues to PRK function. RhoB recruits PRK1 to endosomes and has been suggested to regulate trafficking in HeLa cells (14
). PRK2 has been implicated in the regulation of cadherin-dependent cell-cell adhesion in keratinocytes, as overexpression of PRK2 resulted in increased localization of E-cadherin at cell-cell contacts and increased adhesiveness (7
). In this study, we demonstrate that PRK2 is required for the transition from primordial junctions to mature apical junctions. 16HBE cells depleted of PRK2 were able to form primordial junctions, consisting of punctate E-cadherin complexes at nascent cell-cell contacts, but these primordial junctions did not mature into apical junctions, consisting of tight and adherens junctions and the associated perijunctional actin filaments. We previously showed that PRK2 localizes to the midbody during cytokinesis and that PRK2 depletion in HeLa cells results in defective cell division, leading to the formation of multinucleated cells (35
). No significant increase in multinucleated cells was observed in 16HBE cells depleted of PRK2, although PRK2 does localize at the midbody of these cells during cytokinesis (not shown).
PRK2 localizes to primordial junctions in 16HBE cells to promote their maturation. Localization of PRK2 at junctions is dependent on its C2-like domain. C2 domains are calcium-dependent phospholipid binding domains first described in classical PKC family members (22
). The C2 domain of PRK lacks critical residues for calcium binding and is therefore referred to as a C2-like domain (30
). C2-like domains might function as calcium-independent phospholipid binding domains but could also function as protein-protein interaction domains (22
). PRK1 interacts with the actin filament binding protein α-actinin, and the interacting region has been mapped to residues 136 to 474, a region that overlaps with the C2-like domain (25
). Interestingly, α-actinin localizes to adherens junctions in epithelial cells, raising the possibility that α-actinin recruits PRK2 to junctions (28
). However, we found that during apical junction formation, α-actinin associates with cortical actin filaments and that it is only found at the cell-cell contact in mature junctions (not shown), so it is unlikely that α-actinin is responsible for recruiting PRK2 to primordial junctions.
Mutations in the HR1 domains of PRK2 to prevent binding to RhoA significantly reduced the junctional localization of PRK2, showing that RhoA binding contributes to this localization. Fluorescence resonance energy transfer (FRET)-based biosensors have been used to study RhoA activation in epithelial cells as junctions form, and it was found that RhoA is activated at nascent cell-cell contacts (46
). The Rho binding-defective mutant did, however, show partial junctional localization in approximately 20% of cells, and yet, it completely failed to rescue tight junction formation after knockdown of endogenous PRK2, suggesting that RhoA is also likely to regulate PRK2 function through an additional mechanism. In vitro
GTP-bound RhoA enhances the kinase activity of PRK2 (41
). The binding of RhoA to the N-terminal GTPase-binding domain of PRK1 has been proposed to disrupt an autoinhibitory closed conformation between the N terminus and the kinase domain (18
). By analogy, the interaction between active RhoA and PRK2 might allow PRK2 to adopt an open conformation at cell-cell contacts, thus activating the kinase domain and facilitating an interaction between the C2-like domain and as-yet-unidentified proteins/lipids to stably localize PRK2 at maturing junctions.
We recently described two additional serine-threonine kinases required for the formation of mature apical junctions in 16HBE cells, PAK4 and aPKC, both Cdc42 targets (42
). Depletion of PRK2, PAK4, or aPKC leads to an apparently identical phenotype in which cells undergo the initial step of junction formation to form primordial junctions but these do not mature into apical junctions. This raises the question of why three separate protein kinase activities would be required for junctional maturation to occur. Apical junction formation requires the coordination of several processes, such as trafficking of proteins to the plasma membrane, regulation of junctional protein complexes, and reorganization of the cortical actin cytoskeleton. It therefore seems likely that PRK2, PAK4, and aPKC regulate different aspects of junction formation by phosphorylating distinct substrates. Future work will be aimed at identifying the relevant substrates of these kinases.