Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity,Resistance to Hepatic Steatosis,and Defective Gluconeogenesis

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a^−/− but not in control or Par-1b^−/− mice. The intercrossing of Par-1a^−/− with Par-1b^−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a^−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b^−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.Cellular polarity is a fundamental principle in biology (6, 36, 62). The prototypical protein kinase originally identified as a regulator of polarity was termed partitioning defective (Par-1) due to early embryonic defects in Caenorhabditis elegans (52). Subsequent studies revealed that Par-1 is required for cellular polarity in worms, flies, frogs, and mammals (4, 17, 58, 63, 65, 71, 89). An integral role for Par-1 kinases in multiple signaling pathways has also been established, and although not formally addressed, multifunctionality for individual Par-1 family members is implied in reviews of the list of recognized upstream regulators and downstream substrates (Table ​(Table1).1). Interestingly, for many Par-1 substrates the phosphorylated residues generate 14-3-3 binding sites (25, 28, 37, 50, 59, 61, 68, 69, 78, 95, 101, 103). 14-3-3 binding in turn modulates both nuclear/cytoplasmic as well as cytoplasmic/membrane shuttling of target proteins, thus allowing Par-1 activity to establish intracellular spatial organization (15, 101). The phosphorylation of Par-1 itself promotes 14-3-3 binding, thereby regulating its subcellular localization (37, 59, 101).

TABLE 1.

Multifunctionality of Par-1 polarity kinase pathways^a

Regulator or substrate	Function	Reference(s)
Regulators (upstream function)
LKB1	Wnt signaling, Peutz-Jeghers syndrome, insulin signal transduction, pattern formation	2, 63, 93
TAO1	MEK3/p38 stress-responsive mitogen-activated protein kinase (MAPK) pathway	46
MARKK	Nerve growth factor signaling in neurite development and differentiation	98
aPKC	Ca2+/DAG-independent signal transduction, cell polarity, glucose metabolism	14, 37, 40, 45, 59, 75, 95
nPKC/PKD	DAG-dependent, Ca2+-independent signal transduction (GPCR)	101
PAR-3/PAR-6/aPKC	(−); regulates Par-1, assembly of microtubules, axon-dendrite specification	19
GSK3β	(−); tau phosphorylation, Alzheimer''s dementia, energy metabolism, body patterning	54, 97
Pim-1 oncogene	(−); G2/M checkpoint, effector of cytokine signaling and Jak/STAT(3/5)	5
CaMKI	(−); Ca2+-dependent signal transduction, neuronal differentiation	99
Substrates (downstream function)
Cdc25C	Regulation of mitotic entry by activation of the cdc2-cyclin B complex	25, 72, 78, 103
Class II HDAC	Control of gene expression and master regulator of subcellular trafficking	28, 50
CRTC2/TORC2	Gluconeogenesis regulator via LKB1/AMPK/TORC2 signaling, PPARγ1a coactivator	49
Dlg/PSD-95	Synaptogenesis and neuromuscular junction, tumor suppressor (102)	104
Disheveled	Wnt signaling, translocation of Dsh from cytoplasmic vesicles to cortex	73, 94
KSR1	Regulation of the Ras-MAPK pathway	68, 69
MAP2/4/TAU	Dynamic instability (67, 83) of microtubules, Alzheimer''s dementia (30)	11, 31-33, 47, 70, 96
Mib/Notch	Mind bomb (Mib degradation and repression of Notch signaling results in neurogenesis)	57, 74, 81
Par3/OSKAR/Lgl	Cytoplasmic protein segregation, cell polarity, and asymmetric cell division	7, 10
Pkp2	Desmosome assembly and organization; nuclear shuttling	68, 69
PTPH1	Linkage between Ser/Thr and Tyr phosphorylation-dependent signaling	103
Rab11-FIP	Regulation of endocytosis (23), trafficking of E-cadherin (64)	34

Open in a separate window^aLKB1 also is known as Par-4; MARKK also is known as Ste20-like; (−), inhibitory/negative regulation has been shown; GPCR, G protein-coupled receptors. MARKK is highly homologous to TAO-1 (thousand-and-one amino acid kinase) (46).The mammalian Par-1 family contains four members (Table ​(Table2).2). Physiological functions of the Par-1b kinase have been studied using targeted gene knockout approaches in mice (9, 44). Two independently derived mouse lines null for Par-1b have implicated this protein kinase in diverse physiological processes, including fertility (9), immune system homeostasis (44), learning and memory (86), the positioning of nuclei in pancreatic beta cells (35, 38), and growth and metabolism (43).

TABLE 2.

Terminology and localization of mammalian Par-1 family members

Synonymsa	Subcellular localization
Par-1a, MARK3, C-TAK1, p78/KP78, 1600015G02Rik, A430080F22Rik, Emk2, ETK-1, KIAA4230, mKIAA1860, mKIAA4230, {"type":"entrez-nucleotide","attrs":{"text":"M80359","term_id":"189511"}}M80359	Basolateralb/apicalc
Par-1b, EMK, MARK2, AU024026, mKIAA4207	Basolateral
Par1c, MARK1	Basolateral
Par1d, MARK4, MARKL1	Not asymmetricd

Open in a separate window^aPar should not to be confused with protease-activated receptor 1 (PAR1 29]); C-TAK1, Cdc twenty-five C-associated kinase 1; MARK, microtubule affinity regulating kinase; MARKL, MAP/microtubule affinity-regulating kinase-like 1.^bBasolateral to a lesser degree than Par-1b (37).^cHuman KP78 is asymmetrically localized to the apical surface of epithelial cells (76).^dVariant that does not show asymmetric localization in epithelial cells when overexpressed (95).Beyond Par-1b, most information regarding the cell biological functions of the Par-1 kinases comes from studies of Par-1a. Specifically, Par-1a has been implicated in pancreatic (76) and hepatocarcinogenesis (51), as well as colorectal tumors (77), hippocampal function (100), CagA (Helicobacter pylori)-associated epithelial cell polarity disruption (82), and Peutz-Jeghers syndrome (48), although the latter association has been excluded recently (27). As a first step toward determining unique and redundant functions of Par-1 family members, mice disrupted for a second member of the family (Par-1a/MARK3/C-TAK1) were generated. We report that Par-1a^−/− mice are viable and develop normally, and adult mice are hypermetabolic, have decreased white and brown adipose tissue mass, and unaltered glucose/insulin handling. However, when challenged by a high-fat diet (HFD), Par-1a^−/− mice exhibit resistance to hepatic steatosis, resistance to glucose intolerance, and the delayed onset of obesity relative to that of control littermates. Strikingly, overnight starvation results in a complete depletion of glycogen and lipid stores along with an increase in autophagic vacuoles in the liver of Par-1a^−/− but not Par-1b^−/− mice. Correspondingly, Par-1a^−/− mice develop hypoketotic hypoglycemia. These findings reveal unique metabolic functions of two Par-1 family members.