Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles.

Abstract

Abstract. Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membranebound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabeled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32p incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.

Interaction of the DYNLT (TCTEX1/RP3) Light Chains and the Intermediate Chains Reveals Novel Intersubunit Regulation during Assembly of the Dynein Complex

“…Antibodies-The antibodies used were 74.1, a mouse monoclonal antibody that reacts with all isoforms of the intermediate chain (42); CT199, a rabbit polyclonal antibody that reacts with all the DYNLRB family members, and R4058, a rabbit polyclonal antibody that reacts with all the DYNLL family members (43,44); HC8, a rabbit polyclonal antibody that reacts with the cytoplasmic dynein heavy chain; and R1B2, a rabbit polyclonal antibody that reacts with the isoforms of the LIC subunit, both from Dr. R. Vallee (23); and anti-HA (12C5) and anti-Myc (9E10) antibodies obtained from the Lymphocyte Culture Center, University of Virginia.…”

Distinct but Overlapping Sites within the Cytoplasmic Dynein Heavy Chain for Dimerization and for Intermediate Chain and Light Intermediate Chain Binding

“…The ICs also function in targeting, so it is possible that the LICs are alternative to the ICs and that only one of the subunits would be present in a dynein molecule. Previous studies (12,13) have demonstrated that anti-IC immunoprecipitations co-precipitate polypeptides of 50 -55 kDa, the identities of which have not been conclusively determined.…”

Circulation and axonal transport in the optic nerve

“…It uses dynein as the molecular motor (Figure 3). 44,45 Dynein is first synthesised in the cell body, transported to the axon tip (the 'positive' end) by both fast and slow anterograde transport systems, 46 and then activated 47 to commence retrograde transport toward the cell body. The structure of the dynein has been well characterised.…”