Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules.
The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.
Ranganathan
Sripriya
S
Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Liu
Chun-Xiang
CX
Migliorini
Mary M
MM
Von Arnim
Christine A F
CA
Peltan
Ithan D
ID
Mikhailenko
Irina
I
Hyman
Bradley T
BT
Strickland
Dudley K
DK
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry*; D009154:Mutation; D019606:Phosphoamino Acids / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C454119:GULP1 protein, human; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D019606:Phosphoamino Acids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000409:Alanine