Biochemical and Structural Characterization of the Pak1-LC8 Interaction

Abstract

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser 88 inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser 88. Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.The dynein light chain, LC8 (DYNLL1), is a highly conserved, small homodimeric protein that is associated with the dynein motor complex through its interaction with the dynein intermediate chain (IC) 4 (1). In addition to dynein, LC8 binds a diverse set of proteins, including the signaling molecules nNOS and Pak1; the apoptosis regulator Bim/Bmf; the transcription factors TRPS1, NRF1, Swallow, and Ciz1; and viral products, including the rabies phosphoprotein, African swine virus, and herpes simplex virus (2, 3). Due to its association with the dynein complex, LC8 is frequently considered to be an adaptor protein linking these nondynein targets to the motor complex for retrograde transport along microtubules. However, recent structural and thermodynamic studies do not support this mechanism (4). Rather, LC8 may directly regulate functional characteristics of interacting proteins independently of its association with the dynein IC. In support of this contention, LC8 inhibits nitric oxide production when bound to nNOS (5, 6) and reduces the affinity of TRPS1 binding to GATA consensus sequences (7). LC8 is also necessary for early transcription of the rabies genome in N2A cells (8). Additional studies indicate that the interaction of LC8 with the rabies phosphoprotein facilitates its nuclear import (9). However, the molecular mechanisms by which LC...

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

“…This narrow range of affinities could hardly determine the specific binding of DYNLL2 when more partners are present simultaneously. The hierarchy of binding affinities proposed in a previous study (26) is corroborated by our data except that myoVa binds five times stronger than Pak1. We conclude that there is a continuum of binding affinities of the various partners irrespective of the putative type of motifs.…”

“…Thus, the observed dissociation constants of monomeric peptides may not be used directly to describe the interaction with dimeric partners, which in fact are bivalent protein ligands (15). Accordingly, we have previously reported an affinity enhancement of dimeric myoVa fragments binding to DYNLL2, compared with a monomeric peptide (2), and the same was noted for a dimeric DIC fragment (26). However, the quantitative relationship between the affinity and the monomer-dimer state of the binding partner was not investigated in previous studies.…”

“…Phosphorylation of Ser-88 of DYNLL could be another way of regulation by shifting the monomer-dimer equilibrium strongly to the monomer state, thus eliminating the binding grooves (29,33). It is not clear which kinase is involved in this regulation; Pak1 was originally shown to phosphorylate DYNLL (10,34); however, a recent study did not support its direct regulatory role (26).…”

“…nNOS), and noncanonical (e.g. myoVa and Pak1) (2,14,(25)(26)(27). However, the functional relevance of this classification has never been investigated.…”

Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin

“…The structures of the monomeric [3] and dimeric [4] forms have been solved, though only the dimeric form is able to bind cargo. Many of the protein cargos have been identified and several complex structures have been reported with peptides derived from their mapped binding sequences [4][5][6]. Despite the variability of their binding motifs all these complexes share a common interaction mode that involves the concave groove at each side of the facing monomer subunits with the peptide acquiring an extended conformation that enlarges the central b-sheet.…”