Serine 88 Phosphorylation of the 8-kDa Dynein Light Chain 1 Is a Molecular Switch for Its Dimerization Status and Functions

Abstract

Dynein light chain 1 (DLC1, also known as DYNLL1, LC8, and PIN), a ubiquitously expressed and highly conserved protein, participates in a variety of essential intracellular events. Transition of DLC1 between dimer and monomer forms might play a crucial role in its function. However, the molecular mechanism(s) that control the transition remain unknown. DLC1 phosphorylation on Ser 88 by p21-activated kinase 1 (Pak1), a signaling nodule, promotes mammalian cell survival by regulating its interaction with Bim and the stability of Bim. Here we discovered that phosphorylation of Ser 88 , which juxtapose each other at the interface of the DLC dimer, disrupts DLC1 dimer formation and consequently impairs its interaction with Bim. Overexpression of a Ser 88 phosphorylation-inactive DLC1 mutant in mammary epithelium cells and in a transgenic animal model caused apoptosis and accelerated mammary gland involution, respectively, with increased Bim levels. Structural and biophysical studies suggested that phosphorylation-mimicking mutation leads to dissociation of the DLC1 dimer to a pure folded monomer. The phosphorylation-induced DLC1 monomer is incapable of binding to its substrate Bim. These findings reveal a previously unrecognized regulatory mechanism of DLC1 in which the Ser 88 phosphorylation acts as a molecular switch for the transition of DLC1 from dimer to monomer, thereby modulating its interaction with substrates and consequently regulating the functions of DLC1.Dyneins are massive minus-to-plus end microtubules motor complexes. Dyneins are categorized into axonemal dyneins and cytoplasmic dyneins on the basis of structural and functional features (1). Cytoplasmic dyneins are essential for a variety of fundamental intracellular events, such as organization and orientation of the mitotic spindle, nuclear migration, Golgi dynamics, retrograde neuronal axonal transport, and trafficking of vesicles and molecules (2-4). Dynein light chain 1 (DLC1) 4 (also known as DYNLL1, LC8, DLC8, and PIN), a ubiquitously expressed 89-amino acid protein, was initially identified as a light chain of the Chlamydomonas outer dynein arm (5). DLC1 is highly conserved from nematodes to mammals, and DLC1 orthologues share more than 90% sequence identity (6). DLC1 binds to a diverse array of proteins and RNAs, including neuronal nitric-oxide synthase (7), IB␣ (8), p53-binding protein 1 (9), GKAP (10), gephyrin postsynaptic scaffolding proteins (11), Bim (12), Swallow (13), estrogen receptor (14), KIBRA (15), CDK2 (16), virus proteins (17, 18), parathyroid hormone mRNA (19), and possibly other proteins (20, 21).Although DLC1 has been shown to bind to many partners, its physiological roles and the upstream regulators of DLC1 remain poorly understood. DLC1 is presumed to have essential cell functions because of its extraordinary sequence conservation across species and ubiquitous expression. Genetic studies in Aspergillus suggested that DLC1 is important for activities of dynein, because a DLC1 temperature-sensitive mutation led to multiple d...

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

“…Binding of the partners to DYNLL could be regulated by phosphorylation of Thr or Ser residues within the DYNLL-binding motif (32). 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).…”

“…Binding Properties of the S88E DYNLL2 Mutant-It has been previously shown that phosphorylation of DYNLL at Ser-88 could inhibit partner binding (10) by promoting dissociation of DYNLL dimers to monomers (29,33). The monomer-dimer equilibrium of the phosphomimetic DYNLL2 S88E mutant is strongly shifted toward the monomer state (29,33).…”

“…It is noteworthy that the ϳ50-fold weaker K d value in case of the mutant mainly resulted from a change in the observed on-rate constant, whereas the off-rate constant was almost unchanged. The monomeric form of the S88E mutant is unable to bind its partners because the binding groove formation is coupled to dimerization (29,33). Because only the dimeric DYNLL is functional in binding, complex formation depletes the small amount of dimers in equilibrium with monomers in the mutant.…”

“…The monomer-dimer equilibrium of the phosphomimetic DYNLL2 S88E mutant is strongly shifted toward the monomer state (29,33). Binding affinities of the wild type and the mutant DYNLL2 were determined using a fluorescein-labeled peptide corresponding to the binding motif of Bmf by a fluorescence anisotropy titration assay (Table 2 and Fig.…”

Structural Analysis of the Regulation of the DYNLL/LC8 Binding to Nek9 by Phosphorylation

“…Signaling inputs such as LC8 phosphorylation, pH variations, or the cellular redox state have been shown to regulate LC8 function by controlling its dimerization (28 -30). Interestingly, it has been recently shown that phosphorylation of Ser 88 in LC8, which promotes the disruption of the LC8 homodimer, can regulate the binding of protein partners such as dynein intermediate chain (27,31).…”