Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules.
Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association.
Albany Medical Center, Center for Cardiovascular Sciences, ME 418, 47 New Scotland Av., Albany, NY 12208, USA.
Josefa
J
Andrade
Sandy Timm
ST
Pearce
Hu
H
Zhao
Margarida
M
Barroso
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001921:Brain; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003593:Cytoplasm / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008074:Lipoproteins / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology; D008861:Microsomes / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D008074:Lipoproteins; D008869:Microtubule-Associated Proteins; D009226:Myristates; D010455:Peptides; C098929:calcium binding protein p22, rat; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases