Potential Role for Phosphorylation in Differential Regulation of the Assembly of Dynein Light Chains

Abstract

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.Cytoplasmic dynein is a principal cellular motor responsible for minus end directed traffic along microtubules. It is a large multisubunit protein complex involved in a variety of processes, including mitotic spindle assembly and orientation, chromosome segregation, intracellular trafficking of vesicles and mRNA, and the establishment of cell polarity (reviewed in Ref. (1)). The heavy chain subunits of dynein provide ATPase and microtubule binding functions, whereas the intermediate and light chain subunits are thought to mediate binding and transport of a wide array of cargo. LC8 2 and Tctex-1, two of the three light chain subunits of cytoplasmic dynein, tightly associate with, and increase the structure of, disordered dynein intermediate chain (IC) (2, 3). The association with either dimeric light chain is postulated to be essential for dimerization of the intermediate chain (4), although recent work has challenged this hypothesis (5). In the cell, significant amounts of LC8 and Tctex-1 are not incorporated into the dynein complex, raising the possibility that these proteins have additional roles independent of dynein (6, 7). Consistent with this hypothesis, several proteins unrelated to dynein bind LC8 and Tctex-1 (8 -20). Many of these interactions are interpreted to indicate that these molecules are cargoes transported by dynein, and except for the Swallow protein which is required for the proper localization of bicoid mRNA (14), there is little evidence for their active transport along microtubules by dynein. The question remains as to how these ubiquitously expressed highly conserved light chains are regulated for multiple functional roles both as part of dynein and as part of other com...

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).…”

“…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.…”

“…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.…”

Phosphorylation of DYNLT1 at Serine 82 Regulates Microtubule Stability and Mitochondrial Permeabilization in Hypoxia

“…DYNLT1's structure is highly conservative and little is known about its modifications with the main thought to be phosphorylation (Song et al, 2007). Within a protein, phosphorylation can occur on several amino acids and one of the most common phosphorylation states occurs on serine residues.…”