4 research outputs found
Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development
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
Resonance assignments and secondary structure analysis of dynein light chain 8 by magic-angle spinning NMR spectroscopy
Biochemical and Structural Characterization of the Pak1-LC8 Interaction*S⃞
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 Ser88 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 Ser88. 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