Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development

Abstract

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Mutually Exclusive Cytoplasmic Dynein Regulation by NudE-Lis1 and Dynactin

“…The LC8 binding sequence does not contain the canonical LC8 binding motif identified in many other LC8 interactors (53), but whether this indicates a unique mode of LC8 binding is uncertain (Refs. 54,55). We also found the NudE-dynein interaction to be unaffected by exposure of NudE to LC8 before, during, or after dynein-NudE binding (Figs.…”

Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells

“…It is interesting that nuclear translocation of PAK1 was also observed across squamous tumor subtypes, including NSCLC (25%; n = 67) and HNSCC (42%; n = 130) (Tables S2-S6). The biological function of PAK1 in the nucleus is presently not well-understood; however, it has been shown that nuclear import of PAK1 can play a critical role in vertebrate development (36). Loss of function studies to analyze the role of PAK1 in squamous NSCLC cells revealed a requirement for PAK1 in cytoskeletal reorganization, including paxillin phosphorylation, focal adhesion turnover, and cell motility (Fig.…”

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

“…Based on our quantitative analysis of complex formation of representative members of the DYNLL binding partners as monovalent and bivalent ligands, it is likely that at physiological protein concentrations (mostly submicromolar to micromolar in range) (53) the DYNLL-binding sites would be saturated only by dimeric interacting proteins. Indeed, most of the known binding partners of DYNLL are either dimeric or promoted to form dimers upon DYNLL binding by physically holding the two interacting polypeptide chains close to each other (54). Higher apparent affinity of DYNLL binding to dimeric protein fragments was first noted by studying DYNLL2 and myoVa interactions (2) and later attributed to avidity (15).…”