Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor protein KIF21A

Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18788-93. doi: 10.1073/pnas.0810104105. Epub 2008 Nov 19.

Abstract

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates human ADP-ribosylation factor (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Liquid chromatography MS/MS analysis of peptides from proteins that co-precipitated with BIG1 antibodies identified "kinesin family member 21A" (KIF21A), a plus-end-directed motor protein that moves cargo on microtubules away from the microtubule-organizing center. Reciprocal immunoprecipitation (IP) of endogenous proteins and microscopically apparent overlap of immunoreactive BIG1 with overexpressed GFP-KIF21A in the perinuclear region were consistent with an interaction of KIF21A-BIG1. Overexpression of full-length KIF21A and BIG1 and their fragments in HEK293 cells followed by reciprocal IP revealed that the C-terminal tail of KIF21A, with seven WD-40 repeats, may interact with structure in the C-terminal region of BIG1. Interfering with cyclic activation and inactivation of ARF1 by overexpressing constitutively active ARF1(Q71L) or dominant inactive ARF1(T31N) altered the distribution of BIG1 as well as its interaction with KIF21A. A requirement for ARF1 was confirmed by its selective depletion with siRNA. Unlike disruption of microtubules with nocodazole, selective inhibition of transport by depletion of KIF21A with specific siRNA altered BIG1 distribution without changing that of intrinsic Golgi membrane proteins. These newly recognized interactions of BIG1 and KIF21A should enable us to understand better the mechanisms through which, acting together, they may integrate local events in membrane trafficking with longer-range transport processes and to relate those processes to the diverse signaling and scaffold functions of BIG1.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • ADP-Ribosylation Factor 1 / genetics
  • ADP-Ribosylation Factor 1 / metabolism
  • ADP-Ribosylation Factors / genetics
  • ADP-Ribosylation Factors / metabolism
  • Animals
  • Cell Line
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism
  • Humans
  • Kinesin / genetics
  • Kinesin / metabolism*
  • Microtubules / metabolism
  • Nocodazole / metabolism
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Rabbits
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Tubulin / genetics
  • Tubulin / metabolism
  • Tubulin Modulators / metabolism

Substances

  • ARFGEF1 protein, human
  • Guanine Nucleotide Exchange Factors
  • KIF21A protein, human
  • Peptide Fragments
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Tubulin
  • Tubulin Modulators
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • ARF3 protein, human
  • Kinesin
  • ADP-Ribosylation Factor 1
  • ADP-Ribosylation Factors
  • Nocodazole