The role of kinase activity and the kinase insert region in ligand-induced internalization and degradation of the c-fms protein.

Abstract

Molecular steps in endocytosis and degradation of the c‐fms protein were analyzed by following the fate of mutated c‐fms molecules after M‐CSF binding. A mutant c‐fms protein lacking tyrosine kinase activity was rapidly internalized after M‐CSF binding but not degraded. Another mutant c‐fms molecule that lacked most of the kinase insert region was similarly internalized after M‐CSF binding and also not degraded. This indicates that the signal for internalization is separate from that directing degradation of the receptor. It has been shown previously that a c‐fms mutant in which the kinase insert domain is deleted retains tyrosine kinase activity but lacks two major sites of autophosphorylation. The degradation step therefore requires both kinase activity and the kinase insert region whereas the internalization step is independent of these factors. The major sites of tyrosine autophosphorylation within the kinase insert region were next mutated to determine whether autophosphorylation in the kinase insert region of c‐fms might be the signal that triggers degradation of internalized receptors. These mutant receptors were still rapidly degraded in response to M‐CSF. Therefore, ligand‐induced degradation of c‐fms may require tyrosine phosphorylation of a protein other than the c‐fms receptor itself and the kinase insert region may be necessary for recognition of this substrate.

Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity

“…Therefore these results do not support the previous conclusions that M-CSF-induced degradation is dependent upon receptor autophosphorylation or upon substrate phosphorylation (Downing et al, 1989;Carlberg et al, 1991). These results also raise doubts about some of the conclusions from studies of the downregulation of other receptors that used equivalent lysine mutant receptors (see Introduction) and emphasize the importance of using more than one mutation to test for the dependence of receptor function upon kinase activity.…”

“…Downregulation of the receptors for EGF (Honneger et al, 1987;Glenney et al, 1988), insulin (Russell et al, 1987;McClain et al, 1987;Hari and Roth, 1987), M-CSF (Downing et al, 1989;Carlberg et al, 1991) and PDGF (Sorkin et al, 1991) is thought to be dependent upon intrinsic receptor tyrosine kinase activity, largely because both processes are inhibited by mutation of an invariant lysine residue of the b3 strand ( Figure 1a). In support of this, multiple mutations of tyrosine autophosphorylation sites within the carboxyl terminus of the EGF receptor also inhibit downregulation (Sorkin et al, 1992) as do mutations of autophosphorylation sites within the activating loop of the kinase domain of the insulin receptor (Carpentier, 1994).…”

“…It has been suggested that downregulation of the M-CSF receptor is dependent upon substrate phosphorylation (Carlberg et al, 1991). A di erent model is that autophosphorylation of the EGF receptor stabilizes a change in its structure that exposes cryptic internalization and degradation sites (Opresko et al, 1995).…”

“…Figure 1a shows the single amino acid changes that were separately introduced by oligo-directed mutagenesis to give six di erent kinase-defective M-CSF receptors. One of the receptors has a previously described kinase inactivating mutation of an invariant lysine residue at position 616 (Downing et al, 1989;Carlberg et al, 1991). The sequences of cAPK and the insulin receptor kinase are shown for comparison.…”

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

“…Deletion of the kinase insert region stabilizes the normal receptor (Carlberg et al, 1991). Figure 6b, lane 4 shows that a mutant D802V receptor with a kinase insert deletion has similar steady state levels to a normal M-CSF receptor (Figure 6b, lane 1) and that the immature and mature mutant receptors are smaller due to the deletion.…”