Interaction between LIS1 and PDE4, and its role in cytoplasmic dynein function

Abstract

SummaryLIS1, a WD40 repeat scaffold protein, interacts with components of the cytoplasmic dynein motor complex to regulate dyneindependent cell motility. Here, we reveal that cAMP-specific phosphodiesterases (PDE4s) directly bind PAFAH1B1 (also known as LIS1). Dissociation of LIS1-dynein complexes is coupled with loss of dynein function, as determined in assays of both microtubule transport and directed cell migration in wounded monolayers. Such loss in dynein functioning can be achieved by upregulation of PDE4, which sequesters LIS1 away from dynein, thereby uncovering PDE4 as a regulator of dynein functioning. This process is facilitated by increased intracellular cAMP levels, which selectively augment the interaction of long PDE4 isoforms with LIS1 when they become phosphorylated within their regulatory UCR1 domain by protein kinase A (PKA). We propose that PDE4 and dynein have overlapping interaction sites for LIS1, which allows PDE4 to compete with dynein for LIS1 association in a process enhanced by the PKA phosphorylation of PDE4 long isoforms. This provides a further example to the growing notion that PDE4 itself may provide a signalling role independent of its catalytic activity, exemplified here by its modulation of dynein motor function. Journal of Cell Scienceanti-VSV agarose and immune complexes blotted for both the PDE4 proteins and LIS1-GFP. Specific co-capture of LIS1-GFP was observed in the immunoprecipitates (Fig. 1A).Where indicated, cells were treated with a combination of the adenylyl cyclase activator forskolin and the non-selective cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine 2254 Journal of Cell Science 124 (13) Fig. 1. Interaction of LIS1 with PDE4 isoforms. (A)C-terminal GFP-tagged LIS1 (LIS1-GFP) was co-expressed in HEK293 cells with VSV-epitope-tagged long PDE4 isoforms, either PDE4B1 or PDE4D3. Single transfections of LIS1-GFP, PDE4B1-VSV or PDE4D3-VSV were also performed as controls. Cells were challenged with either vehicle or with 100mM forskolin and 100mM IBMX for 15 minutes prior to harvesting. Lysates were immunoprecipitated with anti-VSV agarose and immunoblotted with anti-LIS1 or anti-VSV antibodies. The relative expression levels of the indicated proteins in cell lysate are shown in the input lanes. (B)HEK293 cells were co-transfected with LIS1 and the GFP-tagged short PDE4B2 isoform. Cells were challenged as described for A and lysates treated with anti-LIS1 antibody. Immunoprecipitates and input lysates were probed with GFP or LIS1 antisera. (C)Non-transfected HEK293 cells were treated as for A and cell lysates treated with PDE4B2-specific or PDE4D5-specific antibodies. Co-precipitated LIS1 was visualised using anti-LIS1 antibody (upper panels). The relative expression levels of LIS1, PDE4D5 and PDE4B2 in cell lysates were determined by specific immunoblotting. (D)HEK293 cells were transfected to coexpress LIS1-GFP with wild-type PDE4D3-VSV (i,ii) or the PDE4D3-VSV mutants, PDE4D3-S54A (i), PDE4D3-S13A (i) and PDE4D3-R51A,R52A (ii). Cell...

The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s)

“…Recent studies suggest that PDE4s may act as scaffolding proteins [10, 40, 41], thus exerting functions independent of their catalytic activity, by mediating the assembly of macromolecular signaling complexes. The ability of PDE4 protomers to engage in independent protein interactions may support scaffolding function of PDEs and hetero-oligomerization may allow for further complexity in protein interactions (see Figure 8).…”

PDE4 as a target for cognition enhancement

“…Developing this idea a step further, several groups mapped the interaction domains between PDE4s and their scaffold proteins and developed disrupting peptides based on this information. Treatment of cells with these peptides was shown to augment discrete pools of cAMP and thereby exert specific biological functions via PDE4 displacement from signaling complexes [109,137,138]. Although no therapeutic has been generated thus far, these studies demonstrate, in proof of concept, that development of small molecule disruptors of PDE4 signaling complexes is a viable option for future drug development.…”

Functional coupling of microtubules to membranes – implications for membrane structure and dynamics

“…It is probable that phospholipid remodelling and motor function are both involved in LIS1-dependent membrane dynamics. An interesting additional aspect of LIS1 function comes from the recent finding that cAMP-specific phosphodiesterase 4 (PDE4) can negatively regulate the association of LIS1 with dynein, thereby providing a potential hub that integrates signalling with LIS1-dependent dynein function (Murdoch et al, 2011). Thus, NDE1 and LIS1 appear to act together to generate a dynein complex that is capable of moving heavy loads, whereas dynactin acts to enhance processivity.…”