The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

Abstract

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

“…Despite a growing body of evidence from yeast and other organisms linking TORC2 to lipids (7-9), a precise link between TORC2 substrates and sphingolipid metabolism had not been established. However, we and others demonstrated that two paralogous protein kinases, Ypk1/Ykl126w and Ypk2/Ymr104c (mammalian orthologs are SGK1, SGK2, and SGK3) (10), now known to be TORC2 targets (11,12), have essential roles in processes linked to membrane dynamics (13)(14)(15). In particular, we had previously implicated Ypk1 function in the regulation of sphingolipid biosynthesis in two ways.…”

Identification of Ypk1 as a Novel Selective Substrate for Nitrogen Starvation-triggered Proteolysis Requiring Autophagy System and Endosomal Sorting Complex Required for Transport (ESCRT) Machinery Components

“…Rapid Ypk1 degradation using an autophagy system may ensure that yeast cells manage translational arrest and possibly autophagic protein breakdown in cellular nitrogen utilization. In addition to protein translation, Ypk1 is also involved in various cellular signaling pathways such as actin regulation and endocytosis (7,8). Cellular nitrogen utilization may control these cellular events.…”

“…Although a direct substrate of this yeast protein kinase has not been identified, loss of Ypk1 causes several cellular deficiencies in actin cytoskeletal organization (7) and endocytosis (8) as well as resistance to ISP-1/myriocin, a potent inhibitor of sphingolipid biosynthesis (9). As a common feature of AGC kinases, Ypk1 kinase activity is regulated by three phosphorylations at the T-loop, turn motif, and hydrophobic motif.…”

Roles of Pdk1p, a Fission Yeast Protein Related to Phosphoinositide-dependent Protein Kinase, in the Regulation of Mitosis and Cytokinesis

“…Although neither is essential, combined deletion leads to loss of viability of cells (Casamayor et al, 1999). Similarly to other members of the family, they phosphorylate and activate effectors belonging to the AGC family of protein kinases, thereby regulating various cellular processes, such as growth, survival, stress response, cell wall integrity, and endocytosis (Friant et al, 2001;deHart et al, 2002;Roelants et al, 2002Roelants et al, , 2004.…”

“…Although neither is essential, combined deletion leads to loss of viability of cells (Casamayor et al, 1999). Similarly to other members of the family, they phosphorylate and activate effectors belonging to the AGC family of protein kinases, thereby regulating various cellular processes, such as growth, survival, stress response, cell wall integrity, and endocytosis (Friant et al, 2001;deHart et al, 2002;Roelants et al, 2002Roelants et al, , 2004.Although several functions have been attributed to Pdk1p-related proteins in metazoans, the finding that Pdk1p-related proteins exist in single-celled organisms raises the possibility that these molecules might be involved in fundamental cellular mechanisms that occur in all cell types. Here, we show that the fission yeast Pdk1p, a component of the mitotic spindle pole bodies (SPBs) and the medial actomyosin ring, is important for progression through mitosis and cytokinesis.…”